Engineered glycosyltransferases and steviol glycoside glucosylation methods

ABSTRACT

The present invention provides engineered glycosyltransferase (GT) enzymes, polypeptides having GT activity, and polynucleotides encoding these enzymes, as well as vectors and host cells comprising these polynucleotides and polypeptides. The present invention provides engineered sucrose synthase (SuS) enzymes, polypeptides having SuS activity, and polynucleotides encoding these enzymes, as well as vectors and host cells comprising these polynucleotides and polypeptides. The present invention also provides compositions comprising the GT enzymes and methods of using the engineered GT enzymes to make products with β-glucose linkages. The present invention further provides compositions and methods for the production of rebaudiosides (e.g., rebaudioside M, rebaudioside A, rebaudioside I, and rebaudioside D). The present invention also provides compositions comprising the SuS enzymes and methods of using them. Methods for producing GT and SuS enzymes are also provided.

The present application is a Divisional of co-pending U.S. patentapplication Ser. No. 15/886,094, filed Feb. 1, 2018, which claimspriority to U.S. Prov. Pat. Appln. Ser. No. 62/454,417, filed Feb. 3,2017, and U.S. Prov. Pat. Appln. Ser. No. 62/479,262, filed Mar. 30,2017, all of which are hereby incorporated by reference in theirentireties, for all purposes.

FIELD OF THE INVENTION

The present invention provides engineered glycosyltransferase (GT)enzymes, polypeptides having GT activity, and polynucleotides encodingthese enzymes, as well as vectors and host cells comprising thesepolynucleotides and polypeptides. The present invention providesengineered sucrose synthase (SuS) enzymes, polypeptides having SuSactivity, and polynucleotides encoding these enzymes, as well as vectorsand host cells comprising these polynucleotides and polypeptides. Thepresent invention also provides compositions comprising the GT enzymesand methods of using the engineered GT enzymes to make products withβ-glucose linkages. The present invention further provides compositionsand methods for the production of rebaudiosides (e.g., rebaudioside M,rebaudioside A, rebaudioside I, and rebaudioside D). The presentinvention also provides compositions comprising the SuS enzymes andmethods of using them. Methods for producing GT and SuS enzymes are alsoprovided.

REFERENCE TO SEQUENCE LISTING, TABLE OR COMPUTER PROGRAM

The official copy of the Sequence Listing is submitted concurrently withthe specification as an ASCII formatted text file via EFS-Web, with afile name of “CX8-162WO3_ST25.txt”, a creation date of Jan. 24, 2018,and a size of 31,100 kilobytes. The Sequence Listing filed via EFS-Webis part of the specification and is incorporated in its entirety byreference herein.

BACKGROUND OF THE INVENTION

Glycosyltransferases (GT) are enzymes that post-translationally transferglycosyl residues from an activated nucleoside sugar to monomeric andpolymeric acceptor molecules (e.g., other sugars, proteins, lipids, andother organic substrates). Thus, these enzymes utilize an activateddonor sugar substrate that contains a substituted phosphate leavinggroup. Donor sugar substrates (i.e., the “glycosyl donor”) are commonlyactivated as nucleoside diphosphate sugars. However, other sugars, suchas nucleoside monophosphate sugars, lipid phosphates and unsubstitutedphosphates are also used (See e.g., Lairson et al., Ann. Rev. Biochem.,77:25.1-25.35 [2008]). GTs are classified as either retaining orinverting enzymes, based on the stereochemistry of the substrates andreaction products. In reactions where the stereochemistry of the donor'sanomeric bond is retained (e.g., alpha to alpha), the GT is a retainingenzyme. In reactions where the stereochemistry is inverted (e.g., alphato beta), the GT is an inverting enzyme. These glycosylated products areinvolved in various metabolic pathways and processes. Indeed, thebiosynthesis of numerous disaccharides, oligosaccharides, andpolysaccharides involve the action of various glycosyltransferases. Thetransfer of a glucosyl moiety can alter the acceptor's bioactivity,solubility, and transport properties within cells. GTs have found use inthe targeted synthesis of specific compounds (e.g., glycoconjugates andglycosides), as well as the production of differentially glycosylateddrug, biological probes or natural product libraries. In some methods,the large scale use of GTs for glycoconjugate synthesis requires largequantities of glycosyl donors, adding to the cost of such approaches.Nucleotide recycling systems have been developed to allow theresynthesis of glycosyl donors from the released nucleotide. Theserecycling systems also reduce the amount of nucleotide by-product formedduring the reaction, thereby reducing inhibition caused by the GT.Nonetheless, the need remains for improved methods suitable forlarge-scale production of glycoconjugates by GTs.

SUMMARY OF THE INVENTION

The present invention provides engineered glycosyltransferase (GT)enzymes, polypeptides having GT activity, and polynucleotides encodingthese enzymes, as well as vectors and host cells comprising thesepolynucleotides and polypeptides. The present invention providesengineered sucrose synthase (SuS) enzymes, polypeptides having SuSactivity, and polynucleotides encoding these enzymes, as well as vectorsand host cells comprising these polynucleotides and polypeptides. Thepresent invention also provides compositions comprising the GT enzymesand methods of using the engineered GT enzymes to make products withβ-glucose linkages. The present invention further provides compositionsand methods for the production of rebaudiosides (e.g., rebaudioside M,rebaudioside A, rebaudioside I, and rebaudioside D). The presentinvention also provides compositions comprising the SuS enzymes andmethods of using them. Methods for producing GT and SuS enzymes are alsoprovided.

The present invention provides engineered glycosyltransferasescomprising polypeptide sequences that have at least 60%, 65%, 70%, 75%,80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99%, or more sequence identity to SEQ ID NO:2. In some embodiments,the engineered glycosyltransferase comprises a polypeptide that has 60%,65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99%, or more sequence identity to SEQ ID NO: 4, 8,32, 232, 348, 548, 562, 696, 758, 770, 792, 954, 1002, 1054, 2600, 2718,2814, 2884, 3016, 3082, 3244, 3346, 3502, 3696, 3956, 4256, 4550, 4684,4838, 4876, 5066, 5290, 5372, 5562, 5708, 5976, 6138, 6288, 6468, 6864,7324, 7388, 7784, and/or 8088. In some additional embodiments, theengineered glycosyltransferase comprises a polypeptide that has at least90% sequence identity to SEQ ID NO: 4, 8, 32, 232, 348, 548, 562, 696,758, 770, 792, 954, 1002, 1054, 2600, 2718, 2814, 2884, 3016, 3082,3244, 3346, 3502, 3696, 3956, 4256, 4550, 4684, 4838, 4876, 5066, 5290,5372, 5562, 5708, 5976, 6138, 6288, 6468, 6864, 7324, 7388, 7784, and/or8088. In some additional embodiments, the engineered glycosyltransferasecomprises a polypeptide that has at least 91% sequence identity to SEQID NO: 4, 8, 32, 232, 348, 548, 562, 696, 758, 770, 792, 954, 1002,1054, 2600, 2718, 2814, 2884, 3016, 3082, 3244, 3346, 3502, 3696, 3956,4256, 4550, 4684, 4838, 4876, 5066, 5290, 5372, 5562, 5708, 5976, 6138,6288, 6468, 6864, 7324, 7388, 7784, and/or 8088. In some additionalembodiments, the engineered glycosyltransferase comprises a polypeptidethat has at least 92% sequence identity to SEQ ID NO: 4, 8, 32, 232,348, 548, 562, 696, 758, 770, 792, 954, 1002, 1054, 2600, 2718, 2814,2884, 3016, 3082, 3244, 3346, 3502, 3696, 3956, 4256, 4550, 4684, 4838,4876, 5066, 5290, 5372, 5562, 5708, 5976, 6138, 6288, 6468, 6864, 7324,7388, 7784, and/or 8088. In some additional embodiments, the engineeredglycosyltransferase comprises a polypeptide that has at least 93%sequence identity to SEQ ID NO: 4, 8, 32, 232, 348, 548, 562, 696, 758,770, 792, 954, 1002, 1054, 2600, 2718, 2814, 2884, 3016, 3082, 3244,3346, 3502, 3696, 3956, 4256, 4550, 4684, 4838, 4876, 5066, 5290, 5372,5562, 5708, 5976, 6138, 6288, 6468, 6864, 7324, 7388, 7784, and/or 8088.In some additional embodiments, the engineered glycosyltransferasecomprises a polypeptide that has at least 94% sequence identity to SEQID NO: 44, 8, 32, 232, 348, 548, 562, 696, 758, 770, 792, 954, 1002,1054, 2600, 2718, 2814, 2884, 3016, 3082, 3244, 3346, 3502, 3696, 3956,4256, 4550, 4684, 4838, 4876, 5066, 5290, 5372, 5562, 5708, 5976, 6138,6288, 6468, 6864, 7324, 7388, 7784, and/or 8088. In some additionalembodiments, the engineered glycosyltransferase comprises a polypeptidethat has at least 95% sequence identity to SEQ ID NO: 4, 8, 32, 232,348, 548, 562, 696, 758, 770, 792, 954, 1002, 1054, 2600, 2718, 2814,2884, 3016, 3082, 3244, 3346, 3502, 3696, 3956, 4256, 4550, 4684, 4838,4876, 5066, 5290, 5372, 5562, 5708, 5976, 6138, 6288, 6468, 6864, 7324,7388, 7784, and/or 8088. In some additional embodiments, the engineeredglycosyltransferase comprises a polypeptide that has at least 96%sequence identity to SEQ ID NO: 4, 8, 32, 232, 348, 548, 562, 696, 758,770, 792, 954, 1002, 1054, 2600, 2718, 2814, 2884, 3016, 3082, 3244,3346, 3502, 3696, 3956, 4256, 4550, 4684, 4838, 4876, 5066, 5290, 5372,5562, 5708, 5976, 6138, 6288, 6468, 6864, 7324, 7388, 7784, and/or 8088.In some additional embodiments, the engineered glycosyltransferasecomprises a polypeptide that has at least 97% sequence identity to SEQID NO: 4, 8, 32, 232, 348, 548, 562, 696, 758, 770, 792, 954, 1002,1054, 2600, 2718, 2814, 2884, 3016, 3082, 3244, 3346, 3502, 3696, 3956,4256, 4550, 4684, 4838, 4876, 5066, 5290, 5372, 5562, 5708, 5976, 6138,6288, 6468, 6864, 7324, 7388, 7784, and/or 8088. In some additionalembodiments, the engineered glycosyltransferase comprises a polypeptidethat has at least 98% sequence identity to SEQ ID NO: 44, 8, 32, 232,348, 548, 562, 696, 758, 770, 792, 954, 1002, 1054, 2600, 2718, 2814,2884, 3016, 3082, 3244, 3346, 3502, 3696, 3956, 4256, 4550, 4684, 4838,4876, 5066, 5290, 5372, 5562, 5708, 5976, 6138, 6288, 6468, 6864, 7324,7388, 7784, and/or 8088. In some additional embodiments, the engineeredglycosyltransferase comprises a polypeptide that has at least 99%sequence identity to SEQ ID NO: 44, 8, 32, 232, 348, 548, 562, 696, 758,770, 792, 954, 1002, 1054, 2600, 2718, 2814, 2884, 3016, 3082, 3244,3346, 3502, 3696, 3956, 4256, 4550, 4684, 4838, 4876, 5066, 5290, 5372,5562, 5708, 5976, 6138, 6288, 6468, 6864, 7324, 7388, 7784, and/or 8088.In some additional embodiments, the engineered glycosyltransferasecomprises a polypeptide selected from SEQ ID NO: 4, 8, 32, 232, 348,548, 562, 696, 758, 770, 792, 954, 1002, 1054, 2600, 2718, 2814, 2884,3016, 3082, 3244, 3346, 3502, 3696, 3956, 4256, 4550, 4684, 4838, 4876,5066, 5290, 5372, 5562, 5708, 5976, 6138, 6288, 6468, 6864, 7324, 7388,7784, and/or 8088. In some additional embodiments, the engineeredglycosyltransferase is selected from beta-1,2-glycosyltransferases andbeta-1,3-glycosyltransferases. In some further embodiments, theengineered glycosyltransferase preferentially uses a sugar donor otherthan uracil-diphosphate. In some additional embodiments, the polypeptidesequence of the engineered glycosyltransferase comprises a variantengineered glycosyltransferase provided in Table 2.1, 3.1, 5.1, 6.1,6.3, 8.1, 9.1, 9.2, 9.4, 11.1, 12.1, 14.1, 15.1, 15.2, 15.3, 16.1, 17.1,43.1, 43.2, 44.2, 45.1, 45.3, 46.1, 46.2, 46.3, 47.1, 47.2, 47.3, 48.1,48.2, 49.1, 49.3, 50.1, 50.2, 50.3, 50.4, 51.1, 51.2, 52.1, 53.1, 53.3,54.1, 54.2, 54.3, 55.1, 55.2, 55.3, 56.1, 56.2, 56.3, 57.1, 58.1, 58.2,58.3, 59.1, 59.3, 59.3, 60.1, 60.2, 61.1, 61.2, 62.1, 62.2, 63.1, 63.2,64.1, 64.2, 65.1, 65.2, 66.1, 66.2, 67.1, 67.2, 67.3, 68.1, 68.2, 69.1,69.2, 70.1, 70.2, 71.1, 71.2, 71.3, 72.1, 72.2, 72.3, 73.1, 73.2, 74.1,74.2, 74.3, 75.1, 75.2, 75.3, 77.1, and/or 77.2. The present inventionprovides engineered glycosyltransferases comprising polypeptidesequences that have at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99%, or more sequence identity to SEQ ID NOS: 6, 8, 10, 12, 14, 16,18, 20, 22, 24, 26, 28, 30, 22, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52,54, 56, 58, 60, 62, 64, 66, 68, 70, 76, 78, 80, 82, 84, 86, 88, 90, 92,94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122,124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150,152, 154, 156, 158, 160, 162, 164, 166, 168, 170, 172, 174, 176, 178,180, 182, 184, 186, 188, 190, 192, 194, 196, 198, 200, 202, 204, 206,208, 210, 212, 214, 216, 218, 220, 222, 224, 226, 228, 230, 232, 234,236, 238, 240, 242, 244, 246, 248, 250, 252, 254, 256, 258, 260, 262,264, 266, 268, 270, 272, 274, 276, 278, 280, 282, 284, 286, 288, 290,292, 294, 296, 298, 300, 302, 304, 306, 308, 310, 312, 314, 316, 318,320, 322, 324, 326, 328, 330, 332, 334, 336, 338, 340, 342, 344, 346,348, 350, 352, 354, 356, 358, 360, 362, 364, 366, 368, 370, 372, 374,376, 378, 380, 382, 384, 386, 388, 390, 392, 394, 396, 398, 400, 402,404, 406, 408, 410, 412, 414, 416, 418, 420, 422, 424, 426, 428, 430,432, 434, 436, 438, 440, 442, 444, 446, 448, 450, 452, 454, 456, 458,460, 462, 464, 466, 468, 470, 472, 474, 476, 478, 480, 482, 484, 486,488, 490, 492, 494, 496, 498, 500, 502, 504, 506, 508, 510, 512, 514,516, 518, 520, 522, 524, 526, 528, 530, 532, 534, 536, 538, 540, 542,544, 546, 548, 550, 552, 554, 556, 558, 560, 562, 564, 566, 568, 570,572, 574, 576, 578, 580, 582, 584, 586, 588, 590, 592, 594, 596, 598,600, 602, 604, 606, 608, 610, 612, 614, 616, 618, 620, 622, 624, 626,628, 630, 632, 634, 636, 638, 640, 642, 644, 646, 648, 650, 652, 654,656, 658, 660, 662, 664, 666, 668, 670, 672, 674, 676, 678, 680, 682,684, 686, 688, 690, 692, 694, 696, 698, 700, 702, 704, 706, 708, 710,712, 714, 716, 718, 720, 722, 724, 726, 728, 730, 732, 734, 736, 738,740, 742, 744, 746, 748, 750, 752, 754, 770, 772, 774, 776, 778, 780,782, 784, 786, 788, 790, 792, 794, 796, 798, 800, 802, 804, 806, 808,810, 812, 814, 816, 818, 820, 822, 824, 826, 828, 830, 832, 834, 836,838, 840, 842, 844, 846, 848, 850, 852, 854, 856, 858, 860, 862, 864,866, 868, 870, 872, 874, 876, 878, 880, 882, 884, 886, 888, 890, 892,894, 896, 898, 900, 902, 904, 906, 908, 910, 912, 914, 916, 918, 920,922, 924, 926, 928, 930, 932, 934, 936, 938, 940, 942, 944, 946, 948,950, 952, 954, 956, 958, 960, 962, 964, 966, 968, 970, 972, 974, 976,978, 980, 982, 984, 986, 988, 990, 992, 994, 996, 998, 1000, 1002, 1004,1006, 1008, 1010, 1012, 1014, 1016, 1018, 1020, 1022, 1024, 1026, 1028,1030, 1032, 1034, 1036, 1038, 1040, 1042, 1044, 1046, 1048, 1050, 1052,1054, 1056, 1058, 1060, 1062, 1064, 1066, 1068, 1070, 1072, 1074, 1076,1078, 1290, 1292, 1294, 2596, 2598, 2600, 2602, 2604, 2606, 2608, 2610,2612, 2614, 2616, 2618, 2620, 2622, 2624, 2626, 2628, 2630, 2632, 2634,2636, 2638, 2640, 2642, 2644, 2646, 2648, 2650, 2652, 2654, 2656, 2658,2660, 2662, 2664, 2666, 2668, 2670, 2672, 2674, 2676, 2678, 2680, 2682,2684, 2686, 2688, 2690, 2692, 2694, 2696, 2698, 2700, 2702, 2704, 2706,2708, 2710, 2712, 2714, 2716, 2718, 2720, 2722, 2724, 2726, 2728, 2730,2732, 2734, 2736, 2738, 2740, 2742, 2744, 2746, 2748, 2750, 2752, 2754,2756, 2758, 2760, 2762, 2764, 2766, 2768, 2770, 2772, 2774, 2776, 2778,2780, 2782, 2784, 2786, 2788, 2790, 2792, 2794, 2796, 2798, 2800, 2802,2804, 2806, 2808, 2810, 2812, 2814, 2816, 2818, 2820, 2822, 2824, 2826,2828, 2830, 2832, 2834, 2836, 2838, 2840, 2842, 2844, 2846, 2848, 2850,2852, 2854, 2856, 2858, 2860, 2862, 2864, 2866, 2868, 2870, 2872, 2874,2876, 2878, 2880, 2882, 2884, 2886, 2888, 2890, 2892, 2894, 2896, 2898,2900, 2902, 2904, 2906, 2908, 2910, 2912, 2914, 2916, 2918, 2920, 2922,2924, 2926, 2928, 2830, 2932, 2934, 2936, 2938, 2940, 2942, 2944, 2946,2948, 2950, 2952, 2954, 2956, 2958, 2960, 2962, 2964, 2966, 2968, 2970,2972, 2974, 2976, 2978, 2980, 2982, 2984, 2986, 2988, 2990, 2992, 2994,2996, 2998, 3000, 3002, 3004, 3006, 3008, 3010, 3012, 3014, 3016, 3018,3020, 3022, 3024, 3026, 3028, 3030, 3032, 3034, 3036, 3038, 3040, 3042,3044, 3046, 3048, 3050, 3052, 3054, 3056, 3058, 3060, 3062, 3064, 3066,3068, 3070, 3072, 3074, 3076, 3078, 3080, 3082, 3084, 3086, 3088, 3090,3092, 3094, 3096, 3098, 3100, 3102, 3104, 3106, 3108, 3110, 3112, 3114,3116, 3118, 3120, 3122, 3124, 3126, 3128, 3130, 3132, 3134, 3136, 3138,3140, 3142, 3144, 3146, 3148, 3150, 3152, 3154, 3156, 3158, 3160, 3162,3164, 3166, 3168, 3170, 3172, 3174, 3176, 3178, 3180, 3182, 3184, 3186,3188, 3190, 3192, 3194, 3196, 3198, 3200, 3202, 3204, 3206, 3208, 3210,3212, 3214, 3216, 3218, 3220, 3222, 3224, 3226, 3228, 3230, 3232, 3234,3236, 3238, 3240, 3242, 3244, 3246, 3248, 3250, 3252, 3254, 3256, 3258,3260, 3262, 3264, 3266, 3268, 3270, 3272, 3274, 3276, 3278, 3280, 3282,3284, 3286, 3288, 3290, 3292, 3294, 3296, 3298, 3300, 3302, 3304, 3306,3308, 3310, 3312, 3314, 3316, 3318, 3320, 3322, 3324, 3326, 3328, 3330,3332, 3334, 3336, 3338, 3340, 3342, 3344, 3346, 3348, 3350, 3352, 3354,3356, 3358, 3360, 3362, 3364, 3366, 3368, 3370, 3372, 3374, 3376, 3378,3380, 3382, 3384, 3386, 3388, 3390, 3392, 3394, 3396, 3398, 3400, 3402,3404, 3406, 3408, 3410, 3412, 3414, 3416, 3418, 3420, 3422, 3424, 3426,3428, 3430, 3432, 3434, 3436, 3438, 3440, 3442, 3444, 3446, 3448, 3450,3452, 3454, 3456, 3458, 3460, 3462, 3464, 3466, 3468, 3470, 3472, 3474,3476, 3478, 3480, 3482, 3484, 3486, 3488, 3490, 3492, 3494, 3496, 3498,3500, 3502, 3504, 3506, 3508, 3510, 3512, 3514, 3516, 3518, 3520, 3522,3524, 3526, 3528, 3530, 3532, 3534, 3536, 3538, 3540, 3542, 3544, 3546,3548, 3550, 3552, 3554, 3556, 3558, 3560, 3562, 3564, 3566, 3568, 3570,3572, 3574, 3576, 3578, 3580, 3582, 3584, 3586, 3588, 3590, 3592, 3594,3596, 3598, 3600, 3602, 3604, 3606, 3608, 3610, 3612, 3614, 3616, 3618,3620, 3622, 3624, 3626, 3628, 3630, 3632, 3634, 3636, 3638, 3640, 3642,3644, 3646, 3648, 3650, 3652, 3654, 3656, 3658, 3660, 3662, 3664, 3666,3668, 3670, 3672, 3674, 3676, 3678, 3680, 3682, 3684, 3686, 3688, 3690,3692, 3694, 3696, 3698, 3700, 3702, 3704, 3706, 3708, 3710, 3712, 3714,3716, 3718, 3720, 3722, 3724, 3726, 3728, 3730, 3732, 3734, 3736, 3738,3740, 3742, 3744, 3746, 3748, 3750, 3752, 3754, 3756, 3758, 3760, 3762,3764, 3766, 3768, 3770, 3772, 3774, 3776, 3778, 3780, 3782, 3784, 3786,3788, 3790, 3792, 3794, 3796, 3798, 3800, 3802, 3804, 3806, 3808, 3810,3812, 3814, 3816, 3818, 3820, 3822, 3824, 3826, 3828, 3830, 3832, 3834,3836, 3838, 3840, 3842, 3844, 3846, 3848, 3850, 3852, 3854, 3856, 3858,3860, 3862, 3864, 3866, 3868, 3870, 3872, 3874, 3876, 3878, 3880, 3882,3884, 3886, 3888, 3890, 3892, 3894, 3896, 3898, 3900, 3902, 3904, 3906,3908, 3910, 3912, 3914, 3916, 3918, 3920, 3922, 3924, 3926, 3928, 3930,3932, 3934, 3936, 3938, 3940, 3942, 3944, 3946, 3948, 3950, 3952, 3954,3956, 3958, 3960, 3962, 3964, 3966, 3968, 3970, 3972, 3974, 3976, 3978,3980, 3982, 3984, 3986, 3988, 3990, 3992, 3994, 3996, 3998, 4000, 4002,4004, 4006, 4008, 4010, 4012, 4014, 4016, 4018, 4020, 4022, 4024, 4026,4028, 4030, 4032, 4034, 4036, 4038, 4040, 4042, 4044, 4046, 4048, 4050,4052, 4054, 4056, 4058, 4060, 4062, 4064, 4066, 4068, 4070, 4072, 4074,4076, 4078, 4080, 4082, 4084, 4086, 4088, 4090, 4092, 4094, 4096, 4098,4100, 4102, 4104, 4106, 4108, 4110, 4112, 4114, 4116, 4118, 4120, 4122,4124, 4126, 4128, 4130, 4132, 4134, 4136, 4138, 4140, 4142, 4144, 4146,4148, 4150, 4152, 4154, 4156, 4158, 4160, 4162, 4164, 4166, 4168, 4170,4172, 4174, 4176, 4178, 4180, 4182, 4184, 4186, 4188, 4190, 4192, 4194,4196, 4198, 4200, 4202, 4204, 4206, 4208, 4210, 4212, 4214, 4216, 4218,4220, 4222, 4224, 4226, 4228, 4230, 4232, 4234, 4236, 4238, 4240, 4242,4244, 4246, 4248, 4250, 4252, 4254, 4256, 4258, 4260, 4262, 4264, 4266,4268, 4270, 4272, 4274, 4276, 4278, 4280, 4282, 4284, 4286, 4288, 4290,4292, 4294, 4296, 4298, 4300, 4302, 4304, 4306, 4308, 4310, 4312, 4314,4316, 4318, 4320, 4322, 4324, 4326, 4328, 4330, 4332, 4334, 4336, 4338,4340, 4342, 4344, 4346, 4348, 4350, 4352, 4354, 4356, 4358, 4360, 4362,4364, 4366, 4368, 4370, 4372, 4374, 4376, 4378, 4380, 4382, 4384, 4386,4388, 4390, 4392, 4394, 4396, 4398, 4400, 4402, 4404, 4406, 4408, 4410,4412, 4414, 4416, 4418, 4420, 4422, 4424, 4426, 4428, 4430, 4432, 4434,4436, 4438, 4440, 4442, 4444, 4446, 4448, 4450, 4452, 4454, 4456, 4458,4460, 4462, 4464, 4466, 4468, 4470, 4472, 4474, 4476, 4478, 4480, 4482,4484, 4486, 4488, 4490, 4492, 4494, 4496, 4498, 4500, 4502, 4504, 4506,4508, 4510, 4512, 4514, 4516, 4518, 4520, 4522, 4524, 4526, 4528, 4530,4532, 4534, 4536, 4538, 4540, 4542, 4544, 4546, 4548, 4550, 4552, 4554,4556, 4558, 4560, 4562, 4564, 4566, 4568, 4570, 4572, 4574, 4576, 4578,4580, 4582, 4584, 4586, 4588, 4590, 4592, 4594, 4596, 4598, 4600, 4602,4604, 4606, 4608, 4610, 4612, 4614, 4616, 4618, 4620, 4622, 4624, 4626,4628, 4630, 4632, 4634, 4636, 4638, 4640, 4642, 4644, 4646, 4648, 4650,4652, 4654, 4656, 4658, 4660, 4662, 4664, 46664668, 4670, 4672, 4674,4676, 4678, 4680, 4682, 4684, 4686, 4688, 4690, 4692, 4694, 4696, 4698,4700, 4702, 4704, 4706, 4708, 4710, 4712, 4714, 4716, 4718, 4720, 4722,4724, 4726, 4728, 4730, 4732, 4734, 4736, 4738, 4740, 4742, 4744, 4746,4748, 4750, 4752, 4754, 4756, 4758, 4760, 4762, 4764, 4766, 4768, 4770,4772, 4774, 4776, 4778, 4780, 4782, 4784, 4786, 4788, 4790, 4792, 4794,4796, 4798, 4800, 4902, 4804, 4806, 4808, 4810, 4812, 4814, 4816, 4818,4820, 4822, 4824, 4826, 4828, 4830, 4832, 4834, 4836, 4838, 4840, 4842,4844, 4846, 4848, 4850, 4852, 4854, 4856, 4858, 4860, 4862, 4864, 4866,4868, 4870, 4872, 4874, 4876, 4878, 4880, 4882, 4884, 4886, 4888, 4890,4892, 4894, 4896, 4898, 4900, 4902, 4904, 4906, 4908, 4910, 4912, 4914,4916, 4918, 4920, 4922, 4924, 4926, 4928, 4930, 4932, 4934, 4936, 4938,4940, 4942, 4944, 4946, 4948, 4950, 4952, 4954, 4956, 4958, 4960, 4962,4964, 4966, 4968, 4970, 4972, 4974, 4976, 4978, 4980, 4982, 4984, 4986,4988, 4990, 4992, 4994, 4996, 4998, 5000, 5002, 5004, 5006, 5008, 5010,5012, 5014, 5016, 5018, 5020, 5022, 5024, 5026, 5028, 5030, 5032, 5034,5036, 5038, 5040, 5042, 5044, 5046, 5048, 5050, 5052, 5054, 5056, 5058,5060, 5062, 5064, 5066, 5068, 5070, 5072, 5074, 5076, 5078, 5080, 5082,5084, 5086, 5088, 5090, 5092, 5094, 5096, 5098, 5100, 5102, 5104, 5106,5108, 5110, 5112, 5114, 5116, 5118, 5120, 5122, 5124, 5126, 5128, 5130,5132, 5134, 5136, 5138, 5140, 5142, 5144, 5146, 5148, 5150, 5152, 5154,5156, 5158, 5160, 5162, 5164, 5166, 5168, 5170, 5172, 5174, 5176, 5178,5180, 5182, 5184, 5186, 5188, 5190, 5192, 5194, 5196, 5198, 5200, 5202,5204, 5206, 5208, 5210, 5212, 5214, 5216, 5218, 5220, 5222, 5224, 5226,5228, 5230, 5232, 5234, 5236, 5238, 5240, 5242, 5244, 5246, 5248, 5250,5252, 5254, 5256, 5258, 5260, 5262, 5264, 5266, 5268, 5270, 5272, 5274,5276, 5278, 5280, 5282, 5284, 5286, 5288, 5290, 5292, 5294, 5296, 5298,5300, 5302, 5304, 5306, 5308, 5310, 5312, 5314, 5316, 5318, 5320, 5322,5324, 5326, 5328, 5330, 5332, 5334, 5336, 5338, 5340, 5342, 5344, 5346,5348, 5350, 5352, 5354, 5356, 5358, 5360, 5362, 5364, 5366, 5368, 5370,5372, 5374, 5376, 5378, 5380, 5382, 5384, 5386, 5388, 5390, 5392, 5394,5396, 5398, 5400, 5402, 5404, 5406, 5408, 5410, 5412, 5414, 5416, 5418,5420, 5422, 5424, 5426, 5428, 5430, 5432, 5434, 5436, 5438, 5440, 5442,5444, 5446, 5448, 5450, 5452, 5454, 5456, 5458, 5460, 5462, 5464, 5466,5468, 5470, 5472, 5474, 5476, 5478, 5480, 5482, 5484, 5486, 5488, 5490,5492, 5494, 5496, 5498, 5500, 5502, 5504, 5506, 5508, 5510, 5512, 5514,5516, 5518, 5520, 5522, 5524, 5526, 5528, 5530, 5532, 5534, 5536, 5538,5540, 5542, 5544, 5546, 5548, 5550, 5552, 5554, 5556, 5558, 5560, 5562,5564, 5566, 5568, 5570, 5572, 5574, 5576, 5578, 5580, 5582, 5584, 5586,5588, 5590, 5592, 5594, 5596, 5598, 5600, 5602, 5604, 5606, 5608, 5610,5612, 5614, 5616, 5618, 5620, 5622, 5624, 5626, 5628, 5630, 5632, 5634,5636, 5638, 5640, 5642, 5644, 5646, 5648, 5650, 5652, 5654, 5656, 5658,5660, 5662, 5664, 5666, 5668, 5670, 5672, 5674, 5676, 5678, 5680, 5682,5684, 5686, 5688, 5690, 5692, 5694, 5696, 5698, 5700, 5702, 5704, 5706,5708, 5710, 5712, 5714, 5716, 5718, 5720, 5722, 5724, 5726, 5728, 5730,5732, 5734, 5736, 5738, 5740, 5742, 5744, 5746, 5748, 5750, 5752, 5754,5756, 5758, 5760, 5762, 5764, 5766, 5768, 5770, 5772, 5774, 5776, 5778,5780, 5782, 5784, 5786, 5788, 5790, 5792, 5794, 5796, 5798, 5800, 5802,5804, 5806, 5808, 5810, 5812, 5814, 5816, 5818, 5820, 5822, 5824, 5826,5828, 5830, 5832, 5834, 5836, 5838, 5840, 5842, 5844, 5846, 5848, 5850,5852, 5854, 5856, 5858, 5860, 5862, 5864, 5866, 5868, 5870, 5872, 5874,5876, 5878, 5880, 5882, 5884, 5886, 5888, 5890, 5892, 5894, 5896, 5898,5900, 5902, 5904, 5906, 5908, 5910, 5912, 5914, 5916, 5918, 5920, 5922,5924, 5926, 5928, 5930, 5932, 5934, 5936, 5938, 5940, 5942, 5944, 5946,5948, 5950, 5952, 5954, 5956, 5958, 5960, 5962, 5964, 5966, 5968, 5970,5972, 5974, 5976, 5978, 5980, 5982, 5984, 5986, 5988, 5990, 5992, 5994,5996, 5998, 6000, 6002, 6004, 6006, 6008, 6010, 6012, 6014, 6016, 6018,6020, 6022, 6024, 6026, 6028, 6030, 6032, 6034, 6036, 6038, 6040, 6042,6044, 6046, 6048, 6050, 6052, 6054, 6056, 6058, 6060, 6062, 6064, 6066,6068, 6070, 6072, 6074, 6076, 6078, 6080, 6082, 6084, 6086, 6088, 6090,6092, 6094, 6096, 6098, 6100, 6102, 6104, 6106, 6108, 6110, 6112, 6114,6116, 6118, 6120, 6122, 6124, 6126, 6128, 6130, 6132, 6134, 6136, 6138,6140, 6142, 6144, 6146, 6148, 6150, 6152, 6154, 6156, 6158, 6160, 6162,6164, 6166, 6168, 6170, 6172, 6174, 6176, 6178, 6180, 6182, 6184, 6186,6188, 6190, 6192, 6194, 6196, 6198, 6200, 6202, 6204, 6206, 6208, 6210,6212, 6214, 6216, 6218, 6220, 6222, 6224, 6226, 6228, 6230, 6232, 6234,6236, 6238, 6240, 6242, 6244, 6246, 6248, 6250, 6252, 6254, 6256, 6258,6260, 6262, 6264, 6266, 6268, 6270, 6272, 6274, 6276, 6278, 6280, 6282,6284, 6286, 6288, 6290, 6292, 6294, 6296, 6298, 6300, 6302, 6304, 6306,6308, 6310, 6312, 6314, 6316, 6318, 6320, 6322, 6324, 6326, 6328, 6330,6332, 6334, 6336, 6338, 6340, 6342, 6344, 6346, 6348, 6350, 6352, 6354,6356, 6358, 6360, 6362, 6364, 6366, 6368, 6370, 6372, 6374, 6376, 6378,6380, 6382, 6384, 6386, 6388, 6390, 6392, 6394, 6396, 6398, 6400, 6402,6404, 6406, 6408, 6410, 6412, 6414, 6416, 6418, 6420, 6422, 6424, 6426,6428, 6430, 6432, 6434, 6436, 6438, 6440, 6442, 6444, 6446, 6448, 6450,6452, 6454, 6456, 6458, 6460, 6462, 6464, 6466, 6468, 6470, 6472, 6474,6476, 6478, 6480, 6482, 6484, 6486, 6488, 6490, 6492, 6494, 6496, 6498,6500, 6502, 6504, 6506, 6508, 6510, 6512, 6514, 6516, 6518, 6520, 6522,6524, 6526, 6528, 6530, 6532, 6534, 6536, 6538, 6540, 6542, 6544, 6546,6548, 6550, 6552, 6554, 6556, 6558, 6560, 6562, 6564, 6566, 6568, 6570,6572, 6574, 6576, 6578, 6580, 6582, 6584, 6586, 6588, 6590, 6592, 6594,6596, 6598, 6600, 6602, 6604, 6606, 6608, 6610, 6612, 6614, 6616, 6618,6620, 6622, 6624, 6626, 6628, 6630, 6632, 6634, 6636, 6638, 6640, 6642,6644, 6646, 6648, 6650, 6652, 6654, 6656, 6658, 6660, 6662, 6664, 6666,6668, 6670, 6672, 6674, 6676, 6678, 6680, 6682, 6684, 6686, 6688, 6690,6692, 6694, 6696, 6698, 6700, 6702, 6704, 6706, 6708, 6710, 6712, 6714,6716, 6718, 6720, 6722, 6724, 6726, 6728, 6730, 6732, 6734, 6736, 6738,6740, 6742, 6744, 6746, 6748, 6750, 6752, 6754, 6756, 6758, 6760, 6762,6764, 6766, 6768, 6770, 6772, 6774, 6776, 6778, 6780, 6782, 6784, 6786,6788, 6790, 6792, 6794, 6796, 6798, 6800, 6802, 6804, 6806, 6808, 6810,6812, 6814, 6816, 6818, 6820, 6822, 6824, 6826, 6828, 6830, 6832, 6834,6836, 6838, 6840, 6842, 6844, 6846, 6848, 6850, 6852, 6854, 6856, 6858,6860, 6862, 6864, 6866, 6868, 6870, 6872, 6874, 6876, 6878, 6880, 6882,6884, 6886, 6888, 6890, 6892, 6894, 6896, 6898, 6900, 6902, 6904, 6906,6908, 6910, 6912, 6914, 6916, 6918, 6920, 6922, 6924, 6926, 6928, 6930,6932, 6934, 6936, 6938, 6940, 6942, 6944, 6946, 6948, 6950, 6952, 6954,6956, 6958, 6960, 6962, 6964, 6966, 6968, 6970, 6972, 6974, 6976, 6978,6980, 6982, 6984, 6986, 6988, 6990, 6992, 6994, 6996, 6998, 7000, 7002,7004, 7006, 7008, 7010, 7012, 7014, 7016, 7018, 7020, 7022, 7024, 7026,7028, 7030, 7032, 7034, 7036, 7038, 7040, 7042, 7044, 7046, 7048, 7050,7052, 7054, 7056, 7058, 7060, 7062, 7064, 7066, 7068, 7070, 7072, 7074,7076, 7078, 7080, 7082, 7084, 7086, 7088, 7090, 7092, 7094, 7096, 7098,7100, 7102, 7104, 7106, 7108, 7110, 7112, 7114, 7116, 7118, 7120, 7122,7124, 7126, 7128, 7130, 7132, 7134, 7136, 7138, 7140, 7142, 7144, 7146,7148, 7150, 7152, 7154, 7156, 7158, 7160, 7162, 7164, 7166, 7168, 7170,7172, 7174, 7176, 7178, 7180, 7182, 7184, 7186, 7188, 7190, 7192, 7194,7196, 7198, 7200, 7202, 7204, 7206, 7208, 7210, 7212, 7214, 7216, 7218,7220, 7222, 7224, 7226, 7228, 7230, 7232, 7234, 7236, 7238, 7240, 7242,7244, 7246, 7248, 7250, 7252, 7254, 7256, 7258, 7260, 7262, 7264, 7266,7268, 7270, 7272, 7274, 7276, 7278, 7280, 7282, 7284, 7286, 7288, 7290,7292, 7294, 7296, 7298, 7300, 7302, 7304, 7306, 7308, 7310, 7312, 7314,7316, 7318, 7320, 7322, 7324, 7326, 7328, 7330, 7332, 7334, 7336, 7338,7340, 7342, 7344, 7346, 7348, 7350, 7352, 7354, 7356, 7358, 7360, 7362,7364, 7366, 7368, 7370, 7372, 7374, 7376, 7378, 7380, 7382, 7384, 7386,7388, 7390, 7392, 7394, 7396, 7398, 7400, 7402, 7404, 7406, 7408, 7410,7412, 7414, 7416, 7418, 7420, 7422, 7424, 7426, 7428, 7430, 7432, 7434,7436, 7766, 7768, 7770, 7772, 7774, 7776, 7778, 7780, 7782, 7784, 7786,7788, 7790, 7792, 7794, 7796, 7798, 7800, 7812, 7814, 7816, 7818, 7820,7822, 7824, 7826, 7828, 7830, 7832, 7834, 7836, 7838, 7840, 7842, 7844,7846, 7848, 7850, 7852, 7854, 7856, 7858, 7860, 7862, 7864, 7866, 7868,7870, 7872, 7874, 7876, 7878, 7880, 7882, 7884, 7886, 7888, 7890, 7892,7894, 7896, 7898, 7900, 7902, 7904, 7906, 7908, 7910, 7912, 7914, 7916,7918, 7920, 7922, 7924, 7926, 7928, 7930, 7932, 7934, 7936, 7938, 7940,7942, 7944, 7946, 7948, 7950, 7952, 7954, 7956, 7958, 7960, 7962, 7964,7966, 7968, 7970, 7972, 7974, 7976, 7978, 7980, 7982, 7984, 7986, 7988,7990, 7992, 7994, 7996, 7998, 8000, 8002, 8004, 8006, 8008, 8010, 8012,8014, 8016, 8018, 8020, 8022, 8024, 8026, 8028, 8030, 8032, 8034, 8036,8038, 8040, 8042, 8044, 8046, 8048, 8050, 8052, 8054, 8056, 8058, 8060,8062, 8064, 8066, 8068, 8070, 8072, 8074, 8076, 8078, 8080, 8082, 8084,8086, 8088, 8090, 8092, 8094, 8096, 8098, 8100, 8102, 8104, 8106, 8108,8110, 8112, 8114, 8116, 8118, 8120, 8122, 8124, 8126, 8128, 8130, 8132,8134, 8136, 8138, 8140, 8142, 8144, 8146, 8148, 8150, 8152, 8154, 8156,8158, 8160, 8162, 8164, 8166, 8168, 8170, 8172, 8174, 8176, 8178, 8180,8182, 8184, 8186, 8188, 8190, 8192, 8194, 8196, 8198, 8200, 8202, 8204,8206, 8208, 8210, 8212, 8214, 8216, 8218, 8220, 8222, 8224, 8226, 8228,8230, 8232, 8234, 8236, 8238, 8240, 8242, 8244, 8246, 8248, 8250, 8252,8254, 8256, 8258, 8260, 8262, 8264, 8266, 8268, 8270, 8272, 8274, 8276,8278, 8280, 8282, 8284, 8286, 8288, 8290, 8292, 8294, 8296, 8298, 8300,8302, 8304, 8306, 8308, 8310, 8312, 8314, 8316, 8318, 8320, 8322, 8324,8326, 8328, 8330, 8332, 8334, 8336, 8338, 8340, 8342, 8344, 8346, 8348,8350, 8352, 8354, 8356, 8358, 8360, 8362, 8364, 8366, 8368, 8482, 8484,8486, 8488, 8490, 8492, 8494, 8496, 8498, 8500, 8502, 8504, 8506, 8508,8510, 8512, 8514, 8516, 8518, 8520, 8522, 8524, 8526, 8528, 8530, 8532,8534, 8536, 8538, 8540, 8542, 8544, 8546, 8548, 8550, 8552, 8554, 8556,8558, 8560, 8562, 8564, 8566, 8568, 8570, 8572, 8574, 8576, 8578, 8580,8582, 8584, 8586, 8588, 8590, 8592, 8594, 8596, 8598, 8600, 8602, 8604,8606, 8608, 8610, 8612, 8614, 8616, 8618, 8620, 8622, 8624, 8626, 8628,8630, 8632, 8634, 8636, 8638, 8640, 8642, 8644, 8646, 8648, 8650, 8652,8654, 8656, 8658, 8660, 8662, 8664, 8666, 8668, 8670, 8672, 8674, 8676,8678, 8680, 8682, 8684, 8686, 8688, 8690, 8692, 8694, 8696, 8698, 8700,8702, 8704, 8706, 8708, 8710, 8712, 8714, 8716, 8718, 8720, 8722, 8724,8726, 8728, 8730, 8732, 8734, 8736, 8738, 8740, 8742, 8744, 8746, 8748,8750, 8752, 8754, 8756, 8758, 8760, 8762, 8764, 8766, 8768, 8770, 8772,8774, 8776, 8778, 8780, 8782, 8784, 8786, 8788, 8790, 8792, 8794, 8796,9108, 9110, 9112, 9114, 9116, 9118, 9120, 9122, 9124, 9126, 9128, 9130,9132, 9134, 9136, 9138, 9140, 9142, 9144, 9146, 9148, 9150, 9152, 9154,9156, 9158, 9160, 9162, 9164, 9166, 9168, 9170, 9172, 9174, 9176, 9178,9180, 9182, 9184, 9186, 9188, 9190, 9192, 9194, 9196, 9198, 9200, 9202,9204, 9206, 9208, 9210, 9212, 9214, 9216, 9218, 9220, 9222, 9224, 9226,9228, 9230, 9232, 9234, 9236, 9238, and/or 9240.

In some further embodiments, the polypeptide sequence of the engineeredglycosyltransferase comprises at least one mutation or mutation set atone or more positions selected from 10, 10/309, 262,278/284/311/339/360, 283, 307, 309, 339/361, 344/361, and 361, whereinthe positions are numbered with reference to SEQ ID NO:4. In someembodiments, the polypeptide sequence of the engineeredGlycosyltransferase comprises at least one mutation or mutation setselected from 10-/309R, 262K, 262L, 278L/284I/311G/339A/360G, 283T,307V, 309L/N/R/S, 339A/361G, 344I/361G, and 361G, wherein the positionsare numbered with reference to SEQ ID NO:4. In some further embodiments,the polypeptide sequence of the engineered glycosyltransferase comprisesat least one mutation or mutation set selected from R10-/V309R, R262K,R262L, Y278L/T284I/R311G/V339A/N360G, S283T, L307V, V309L/N/R/S,V339A/S361G, V344I/S361G, and S361G, wherein the positions are numberedwith reference to SEQ ID NO:4. In still some additional embodiments, thepolypeptide sequence of the engineered glycosyltransferase comprises asequence at least 90% identical to any of SEQ ID NOS: 6, 8, 10, 12, 14,16, 18, 20, 22, 24, 26, 28, and/or 30. In some further embodiments, thepolypeptide sequence of the engineered glycosyltransferase comprises asequence at least identical to any of SEQ ID NOS: 6, 8, 10, 12, 14, 16,18, 20, 22, 24, 26, 28, and/or 30. In yet some additional embodiments,the polypeptide sequence of the engineered glycosyltransferase comprisesSEQ ID NOS: 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, and/or 30.

The present invention also provides engineered glycosyltransferases,wherein the polypeptide sequences of the engineered glycosyltransferasescomprise at least one mutation or mutation set at one or more positionsselected from 112/172/283/318, 112/261/318, 112/282/283/431, 137/283,137/283/431, 163/318, 261/283/306/337, 261/283/337, 261/337, 269/318,282/283, 282/283/431, 283, 283/306/308/360, 283/306/337/426,283/318/337/360, 283/360, 318, 360, and 431, wherein the positions arenumbered with reference to SEQ ID NO:8. In some embodiments, thepolypeptide sequence of the engineered glycosyltransferase comprises atleast one mutation or mutation set selected from 112S/172S/283Q/318E,112S/261S/318E, 112S/282T/283Q/431E, 137K/283Q, 137K/283Q/431E,163K/318E, 261S/283Q/306V/337F, 261S/283Q/337F, 261S/337S, 269T/318E,282T/283Q, 282T/283Q/431E, 283Q, 283Q/306V/3085/360G,283Q/306V/337S/426V, 283Q/318E/337S/360G, 283Q/360G, 318E, 360G, and431E, wherein the positions are numbered with reference to SEQ ID NO:8.In some further embodiments, the polypeptide sequence of the engineeredglycosyltransferase comprises at least one mutation or mutation setselected from E112S/T172S/T283Q/T318E, E112S/R261S/T318E,E112S/S282T/T283Q/Q431E, N137K/T283Q, N137K/T283Q/Q431E, L163K/T318E,R261S/T283Q/L306V/W337F, R261S/T283Q/W337F, R261S/W337S, Q269T/T318E,S282T/T283Q, S282T/T283Q/Q431E, T283Q, T283Q/L306V/R3085/5360G,T283Q/L306V/W337S/A426V, T283Q/T318E/W337S/S360G, T283Q/5360G, T318E,5360G, and Q431E, wherein the positions are numbered with reference toSEQ ID NO:8. In some additional embodiments, the polypeptide sequence ofthe engineered glycosyltransferase comprises a sequence at least 90%identical to any of SEQ ID NOS: 32, 34, 36, 38, 40, 42, 44, 46, 48, 50,52, 54, 56, 58, 60, 62, 64, 66, 68, and/or 70. In some embodiments, thepolypeptide sequence of the engineered glycosyltransferase comprises asequence at least 95% identical to any of SEQ ID NOS: 32, 34, 36, 38,40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, and/or 70.In some additional embodiments, the polypeptide sequence of theengineered glycosyltransferase comprises SEQ ID NOS: 32, 34, 36, 38, 40,42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, and/or 70.

The present invention also provides engineered glycosyltransferases,wherein the polypeptide sequences of the engineered glycosyltransferasescomprise at least one mutation or mutation set at one or more positionsselected from 4, 6, 22, 64, 74, 84, 87, 97, 106, 110, 112, 137, 139,154, 159, 169, 179, 191, 195, 198, 199, 207, 233, 259, 261, 262, 306,347, 356, 396, 417, 421, 427, and 435, wherein the positions arenumbered with reference to SEQ ID NO:32. In yet some additionalembodiments, the polypeptide sequence of the engineeredglycosyltransferase comprises at least one mutation or mutation setselected from 4P, 6P, 22A/L/H/P, 64P, 74W, 84A/G, 87A/H, 97S,106D/G/S/T, 1105, 112A/P, 137G, 139P, 154A/L/Q/V, 159M/R, 169T, 179V,191R, 195G, 198M/S/V, 199A/D/G/K/Q/S, 207L, 233R, 259Q, 261A/H/P/W,262G, 306V, 347D, 356G, 396R, 417A/R/P, 421V, 427A, and 435Q/R, whereinthe positions are numbered with reference to SEQ ID NO:32. In someembodiments, the polypeptide sequence of the engineeredglycosyltransferase comprises at least one mutation or mutation setselected from K4P, E6P, Q22A/L/H/P, F64P, R74W, L84A/G, M87A/H, A97S,L106D/G/S/T, A1105, E112A/P, N137G, R139P, H154A/L/Q/V, Q159M/R, D169T,S179V, S191R, N195G, I198M/S/V, L199A/D/G/K/Q/S, I207L, I233R, H259Q,R261A/H/P/W, T262G, L306V, G347D, S356G, Y396R, E417A/R/P, Y421V, R427A,and V435Q/R, wherein the positions are numbered with reference to SEQ IDNO:32.

The present invention also provides engineered glycosyltransferases,wherein the polypeptide sequences of the engineered glycosyltransferasescomprise at least one mutation or mutation set at one or more positionsselected from 22/84/87/154/198/199/356, 22/84/87/154/198/199/306/356,22/84/87/154/199/306/356, 22/84/87/154/356,22/84/87/154/198/199/306/356, 22/84/87/154/199/306/356,22/84/87/198/199/306/356, 22/84/87/198/199/356,22/84/87/198/199/306/356, 22/84/87/198/199/356, 22/84/87/199/306/356,22/84/87/199/356, 22/84/154/198/199/207/306, 22/84/154/198/199/306,22/84/154/198/199/356, 22/84/154/198/199/356, 22/84/154/198/199/356,22/84/154/199/356, 22/84/154/199/207, 22/84/154/199/356,22/84/154/207/306/356, 22/84/154/306/356, 22/84/154/198/199/306/356,22/84/154/198/199, 22/84/154/198/199/306/356, 22/84/154/199/306/356,22/84/154/199/356, 22/84/154/199/356, 22/84/198/199/306/356,22/84/199/356, 22/84/207/356, 22/84/356, 22/84/154/198/199/306/356,22/87/154/198/199/356, 22/87/154/199/306/356, 22/87/154/322/356,22/87/154/198/199/356, 22/87/154/199/356, 22/87/154/198/199/207/306/356,22/87/154/199/207/356, 22/87/154/199/356, 22/87/154/199/306/356,22/87/198/199/306/356, 22/87/198/199/306/356, 22/87/198/199/207/356,22/87/198/199/356, 22/87/199/356, 22/87/199/356,22/154/198/199/207/306/356, 22/154/198/199/356, 22/154/199/356,22/154/198/199/306/356, 22/154/199/356, 22/154/199/207/306/356,22/198/199/356, 22/198/199/207/329/356, 22/199/356, 22/207/356, 22/356,and 84/154/198/199, wherein the positions are numbered with reference toSEQ ID NO:32. In some additional embodiments, the polypeptide sequenceof the engineered glycosyltransferase comprises at least one mutation ormutation set selected from 22P/84G/87H/154L/198S/199A/356G,22P/84G/87H/154L/198S/199S/306V/356G, 22P/84G/87H/154L/199K/306V/356G,22P/84G/87H/154L/356G, 22P/84G/87H/154V/198S/199S/306V/356G,22P/84G/87H/154V/199K/306V/356G, 22P/84G/87H/198S/199A/306V/356G,22P/84G/87H/198S/199K/356G, 22P/84G/87H/198S/199S/306V/356G,22P/84G/87H/198S/199S/356G, 22P/84G/87H/199A/306V/356G,22P/84G/87H/199S/356G, 22P/84G/154L/198S/199A/207L/306V,22P/84G/154L/198S/199A/306V, 22P/84G/154L/198S/199A/356G,22P/84G/154L/198S/199K/356G, 22P/84G/154L/198S/199S/356G,22P/84G/154L/199A/356G, 22P/84G/154L/199K/207L, 22P/84G/154L/199S/356G,22P/84G/154L/207L/306V/356G, 22P/84G/154L/306V/356G,22P/84G/154V/198S/199A/306V/356G, 22P/84G/154V/198S/199K,22P/84G/154V/198S/199K/306V/356G, 22P/84G/154V/199A/306V/356G,22P/84G/154V/199A/356G, 22P/84G/154V/199S/356G,22P/84G/198S/199K/306V/356G, 22P/84G/199S/356G, 22P/84G/207L/356G,22P/84G/356G, 22P/84V/154V/198S/199S/306V/356G,22P/87H/154L/198S/199K/356G, 22P/87H/154L/199A/306V/356G,22P/87H/154L/3225/356G, 22P/87H/154V/198S/199K/356G,22P/87H/154V/199S/356G, 22P/87H/154V/198S/199S/207L/306V/356G,22P/87H/154V/199A/207L/356G, 22P/87H/154V/199K/356G,22P/87H/154V/199S/306V/356G, 22P/87H/198S/199A/306V/356G,22P/87H/198S/199K/306V/356G, 22P/87H/198S/199S/207L/356G,22P/87H/198S/199K/356G, 22P/87H/199A/356G, 22P/87H/199K/356G,22P/154L/198S/199A/207L/306V/356G, 22P/154L/198S/199A/356G,22P/154L/199A/356G, 22P/154V/198S/199S/306V/356G, 22P/154V/199A/356G,22P/154V/199K/207L/306V/356G, 22P/198S/199A/356G,22P/198S/199K/207L/329C/356G, 22P/199A/356G, 22P/207L/356G, 22P/356G,and 84G/154L/198S/199K, wherein the positions are numbered withreference to SEQ ID NO:32. In some embodiments, the polypeptide sequenceof the engineered glycosyltransferase comprises at least one mutation ormutation set selected from Q22P/L84G/M87H/H154L/I198S/L199A/S356G,Q22P/L84G/M87H/H154L/I198S/L199S/L306V/S356G,Q22P/L84G/M87H/H154L/L199K/L306V/S356G, Q22P/L84G/M87H/H154L/S356G,Q22P/L84G/M87H/H154V/I198S/L199S/L306V/S356G,Q22P/L84G/M87H/H154V/L199K/L306V/S356G,Q22P/L84G/M87H/I198S/L199A/L306V/S356G,Q22P/L84G/M87H/I198S/L199K/S356G,Q22P/L84G/M87H/I198S/L199S/L306V/S356G,Q22P/L84G/M87H/I198S/L199S/S356G, Q22P/L84G/M87H/L199A/L306V/S356G,Q22P/L84G/M87H/L199S/S356G, Q22P/L84G/H154L/I198S/L199A/I207L/L306V,Q22P/L84G/H154L/I198S/L199A/L306V, Q22P/L84G/H154L/I198S/L199A/S356G,Q22P/L84G/H154L/I198S/L199K/S356G, Q22P/L84G/H154L/I198S/L199S/S356G,Q22P/L84G/H154L/L199A/S356G, Q22P/L84G/H154L/L199K/I207L,Q22P/L84G/H154L/L199S/S356G, Q22P/L84G/H154L/I207L/L306V/S356G,Q22P/L84G/H154L/L306V/S356G, Q22P/L84G/H154V/I198S/L199A/L306V/S356G,Q22P/L84G/H154V/I198S/L199K, Q22P/L84G/H154V/I198S/L199K/L306V/S356G,Q22P/L84G/H154V/L199A/L306V/S356G, Q22P/L84G/H154V/L199A/S356G,Q22P/L84G/H154V/L199S/S356G, Q22P/L84G/I198S/L199K/L306V/S356G,Q22P/L84G/L199S/S356G, Q22P/L84G/I207L/S356G, Q22P/L84G/S356G,Q22P/L84V/H154V/I198S/L199S/L306V/S356G,Q22P/M87H/H154L/I198S/L199K/S356G, Q22P/M87H/H154L/L199A/L306V/S356G,Q22P/M87H/H154L/P322S/S356G, Q22P/M87H/H154V/I198S/L199K/S356G,Q22P/M87H/H154V/L199S/S356G,Q22P/M87H/H154V/I198S/L199S/I207L/L306V/S356G,Q22P/M87H/H154V/L199A/I207L/S356G, Q22P/M87H/H154V/L199K/S356G,Q22P/M87H/H154V/L199S/L306V/S356G, Q22P/M87H/I198S/L199A/L306V/S356G,Q22P/M87H/I198S/L199K/L306V/S356G, Q22P/M87H/I198S/L199S/I207L/S356G,Q22P/M87H/I198S/L199K/S356G, Q22P/M87H/L199A/S356G,Q22P/M87H/L199K/S356G, Q22P/H154L/I198S/L199A/I207L/L306V/S356G,Q22P/H154L/I198S/L199A/S356G, Q22P/H154L/L199A/S356G,Q22P/H154V/I198S/L199S/L306V/S356G, Q22P/H154V/L199A/S356G,Q22P/H154V/L199K/I207L/L306V/S356G, Q22P/I198S/L199A/S356G,Q22P/I198S/L199K/I207L/G329C/S356G, Q22P/L199A/S356G, Q22P/I207L/S356G,Q22P/S356G, and L84G/H154L/I198S/L199K, wherein the positions arenumbered with reference to SEQ ID NO:32. In some further embodiments,the polypeptide sequence of the engineered glycosyltransferase comprisesa sequence at least 90% identical to any of SEQ ID NOS: 76, 78, 80, 82,84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114,116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142,144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166, 168, 170,172, 174, 176, 178, 180, 182, 184, 186, 188, 190, 192, 194, 196, 198,200, 202, 204, 206, 208, 210, 212, 214, 216, 218, 220, 222, 224, 226,228, 230, 232, 234, 236, 238, 240, 242, 244, 246, 248, 250, 252, 254,256, 258, 260, 262, 264, 266, 268, 270, 272, 274, 276, 278, 280, 282,284, 286, 288, 290, 292, 294, 296, 298, 300, 302, 304, 306, 308, 310,312, 314, 316, 318, 320, 322, 324, 326, 328, 330, 332, 334, 336, 338,340, 342, 344, 346, and 1290. In some further embodiments, thepolypeptide sequence of the engineered glycosyltransferase comprises asequence at least 95% identical to any of SEQ ID NOS: 76, 78, 80, 82,84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114,116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142,144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166, 168, 170,172, 174, 176, 178, 180, 182, 184, 186, 188, 190, 192, 194, 196, 198,200, 202, 204, 206, 208, 210, 212, 214, 216, 218, 220, 222, 224, 226,228, 230, 232, 234, 236, 238, 240, 242, 244, 246, 248, 250, 252, 254,256, 258, 260, 262, 264, 266, 268, 270, 272, 274, 276, 278, 280, 282,284, 286, 288, 290, 292, 294, 296, 298, 300, 302, 304, 306, 308, 310,312, 314, 316, 318, 320, 322, 324, 326, 328, 330, 332, 334, 336, 338,340, 342, 344, 346, and 1290. In some further embodiments, thepolypeptide sequence of the engineered glycosyltransferase comprises anyof SEQ ID NOS: 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102,104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130,132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158,160, 162, 164, 166, 168, 170, 172, 174, 176, 178, 180, 182, 184, 186,188, 190, 192, 194, 196, 198, 200, 202, 204, 206, 208, 210, 212, 214,216, 218, 220, 222, 224, 226, 228, 230, 232, 234, 236, 238, 240, 242,244, 246, 248, 250, 252, 254, 256, 258, 260, 262, 264, 266, 268, 270,272, 274, 276, 278, 280, 282, 284, 286, 288, 290, 292, 294, 296, 298,300, 302, 304, 306, 308, 310, 312, 314, 316, 318, 320, 322, 324, 326,328, 330, 332, 334, 336, 338, 340, 342, 344, 346, and 1290.

The present invention further provides engineered glycosyltransferases,wherein the polypeptide sequences of the engineered glycosyltransferasescomprise at least one mutation or mutation set at one or more positionsselected from 64/106/109/112/131/159/233/421/427/431,64/106/109/112/417/421/427/431, 64/106/109/139/179/417/421/427/431,64/106/109/233/347/427/431, 64/106/112/347/417/421,64/106/139/179/233/417/427/431, 64/106/417/421, 64/106/431,64/106/109/112/131/179/417/427/431, 64/106/109/417/421/427/431/439,64/106/112/139/159/179/204/396/417, 64/106/112/159/179/417/421,64/106/204/417/421/427, 64/109/112/139/159/179/417/431,64/109/112/139/417, 64/109/139/233/417/421,64/109/159/179/204/233/417/421, 64/109/417/421, 64/109/417/421,64/139/233/417/427/431/439, 64/139/347/417/421/427/431, 64/417/421/431,106/109/112/131/159/179/417/421,106/109/112/131/159/204/347/417/421/427, 106/109/112/204/347/421/439,106/109/112/261/417/431, 106/109/112/347/427, 106/109/139/427/431,106/109/417/421/427, 106/112/159/204/233/417/421/427/431,106/112/233/417, 106/112/396/417/421,106/139/159/233/347/417/421/427/431, 106/233/421/427, 106/417,106/109/139/347/417/421/427, 106/109, 106/109/139/233/417/421,106/109/139/417, 106/109/233/427/431, 106/112/159/179/204/417/421,106/112/159/179/233/417/421/427/439, 106/131/179/233/421/427/431,106/139/421, 106/347/417/427/431, 109/112/131/159/179/439,109/112/131/159/417/421, 109/112/139/179/417/427,109/112/159/179/417/421, 109/112/159/417/427,109/112/179/204/233/417/421/427, 109/112/179/347/417,109/112/204/233/417, 109/112/204/427, 109/112/233/417/431,109/112/417/421/427, 109/112/417/427/431, 109/131/139/179/261/396/421,109/131/204, 109/139/179/417/421/427, 109/139/179/417/427,109/179/233/421, 109/204/417/431, 109/417/421, 109/417/427/431,112/131/179/204/417/421/427, 112/131/179/347/417/421,112/139/179/204/233/347/427, 112/159/417/421, 112/417/421,131/179/233/417/427, 139/233/417, 159/347/417/421/431, 179/417/421,233/417/421/427/431, and 347/417, wherein the positions are numberedwith reference to SEQ ID NO:232. In some embodiments, the polypeptidesequence of the engineered glycosyltransferase comprises at least onemutation or mutation set at one or more positions selected from64P/106S/109R/112P/131P/159R/233R/421V/427L/431D,64P/106S/109R/112P/417A/421V/427A/431D,64P/106S/109R/139P/179L/417R/421V/427L/431D,64P/106S/109R/233R/347D/427A/431D, 64P/106S/112P/347D/417A/421V,64P/106S/139P/179L/233R/417A/427A/431D, 64P/106S/417R/421V,64P/106S/431D, 64P/106T/109R/112P/131P/179L/417R/427A/431D,64P/106T/109R/417A/421V/427L/431D/439P,64P/106T/112P/139P/159R/179V/204D/396R/417A,64P/106T/112P/159R/179L/417R/421V, 64P/106T/204D/417A/421V/427A,64P/109R/112P/139P/159R/179L/417R/431D, 64P/109R/112P/139P/417A,64P/109R/139P/233R/417R/421V, 64P/109R/159R/179V/204D/233R/417R/421V,64P/109R/417A/421V, 64P/109R/417R/421V,64P/139P/233R/417R/427L/431D/439P, 64P/139P/347D/417R/421V/427L/431D,64P/417R/421V/431D, 106S/109R/112P/131P/159R/179L/417A/421V,106S/109R/112P/131P/159R/204D/347D/417A/421V/427L,106S/109R/112P/204D/347D/421V/439P, 106S/109R/112P/261P/417R/431D,106S/109R/112P/347D/427A, 106S/109R/139P/427A/431D,106S/109R/417R/421V/427L, 106S/112P/159R/204D/233R/417R/421V/427A/431D,106S/112P/233R/417R, 106S/112P/396R/417R/421V,106S/139P/159R/233R/347D/417R/421V/427A/431D, 106S/233R/421V/427A,1065/417A, 106T/109R/139P/347D/417A/421V/427A, 106T/109R,106T/109R/139P/233R/417R/421V, 106T/109R/139P/417R,106T/109R/233R/427A/431D, 106T/112P/159R/179L/204D/417A/421V,106T/112P/159R/179V/233R/417A/421V/427L/439P,106T/131P/179L/233R/421V/427L/431D, 106T/139P/421V,106T/347D/417R/427A/431D, 109R/112P/131P/159R/179V/439P,109R/112P/131P/159R/417R/421V, 109R/112P/139P/179V/417R/427A,109R/112P/159R/179V/417R/421V, 109R/112P/159R/417R/427L,109R/112P/179V/204D/233R/417A/421V/427L, 109R/112P/179V/347D/417R,109R/112P/204D/233R/417R, 109R/112P/204D/427A, 109R/112P/233R/417A/431D,109R/112P/417A/421V/427L, 109R/112P/417A/427A/431D,109R/131P/139P/179V/261P/396R/421V, 109R/131P/204D,109R/139P/179L/417R/421V/427A, 109R/139P/179L/417R/427L,109R/179V/233R/421V, 109R/204D/417R/431D, 109R/417R/421V,109R/417R/427A/431D, 112P/131P/179V/204D/417R/421V/427L,112P/131P/179L/347D/417R/421V, 112P/139P/179V/204D/233R/347D/427L,112P/159R/417R/421V, 112P/417R/421V, 131P/179L/233R/417R/427A,139P/233R/417A, 159R/347D/417A/421V/431D, 179V/417R/421V,233R/417R/421V/427L/431D, and 347D/417R, wherein the positions arenumbered with reference to SEQ ID NO:232. In some embodiments, thepolypeptide sequence of the engineered glycosyltransferase comprises atleast one mutation or mutation set at one or more positions selectedfrom F64P/L106S/L109R/E112P/S131P/Q159R/I233R/Y421V/R427L/Q431D,F64P/L106S/L109R/E112P/E417A/Y421V/R427A/Q431D,F64P/L106S/L109R/R139P/S179L/E417R/Y421V/R427L/Q431D,F64P/L106S/L109R/I233R/G347D/R427A/Q431D,F64P/L106S/E112P/G347D/E417A/Y421V,F64P/L106S/R139P/S179L/I233R/E417A/R427A/Q431D, F64P/L106S/E417R/Y421V,F64P/L106S/Q431D, F64P/L106T/L109R/E112P/S131P/S179L/E417R/R427A/Q431D,F64P/L106T/L109R/E417A/Y421V/R427L/Q431D/K439P,F64P/L106T/E112P/R139P/Q159R/S179V/G204D/Y396R/E417A,F64P/L106T/E112P/Q159R/S179L/E417R/Y421V,F64P/L106T/G204D/E417A/Y421V/R427A,F64P/L109R/E112P/R139P/Q159R/S179L/E417R/Q431D,F64P/L109R/E112P/R139P/E417A, F64P/L109R/R139P/I233R/E417R/Y421V,F64P/L109R/Q159R/S179V/G204D/I233R/E417R/Y421V, F64P/L109R/E417A/Y421V,F64P/L109R/E417R/Y421V, F64P/R139P/I233R/E417R/R427L/Q431D/K439P,F64P/R139P/G347D/E417R/Y421V/R427L/Q431D, F64P/E417R/Y421V/Q431D,L106S/L109R/E112P/S131P/Q159R/S179L/E417A/Y421V,L106S/L109R/E112P/S131P/Q159R/G204D/G347D/E417A/Y421V/R427L,L106S/L109R/E112P/G204D/G347D/Y421V/K439P,L106S/L109R/E112P/R261P/E417R/Q431D, L106S/L109R/E112P/G347D/R427A,L106S/L109R/R139P/R427A/Q431D, L106S/L109R/E417R/Y421V/R427L,L106S/E112P/Q159R/G204D/I233R/E417R/Y421V/R427A/Q431D,L106S/E112P/I233R/E417R, L106S/E112P/Y396R/E417R/Y421V,L106S/R139P/Q159R/I233R/G347D/E417R/Y421V/R427A/Q431D,L106S/I233R/Y421V/R427A, L106S/E417A,L106T/L109R/R139P/G347D/E417A/Y421V/R427A, L106T/L109R,L106T/L109R/R139P/I233R/E417R/Y421V, L106T/L109R/R139P/E417R,L106T/L109R/I233R/R427A/Q431D,L106T/E112P/Q159R/S179L/G204D/E417A/Y421V,L106T/E112P/Q159R/S179V/I233R/E417A/Y421V/R427L/K439P,L106T/S131P/S179L/I233R/Y421V/R427L/Q431D, L106T/R139P/Y421V,L106T/G347D/E417R/R427A/Q431D, L109R/E112P/S131P/Q159R/S179V/K439P,L109R/E112P/S131P/Q159R/E417R/Y421V,L109R/E112P/R139P/S179V/E417R/R427A,L109R/E112P/Q159R/S179V/E417R/Y421V, L109R/E112P/Q159R/E417R/R427L,L109R/E112P/S179V/G204D/I233R/E417A/Y421V/R427L,L109R/E112P/S179V/G347D/E417R, L109R/E112P/G204D/I233R/E417R,L109R/E112P/G204D/R427A, L109R/E112P/I233R/E417A/Q431D,L109R/E112P/E417A/Y421V/R427L, L109R/E112P/E417A/R427A/Q431D,L109R/S131P/R139P/S179V/R261P/Y396R/Y421V, L109R/S131P/G204D,L109R/R139P/S179L/E417R/Y421V/R427A, L109R/R139P/S179L/E417R/R427L,L109R/S179V/I233R/Y421V, L109R/G204D/E417R/Q431D, L109R/E417R/Y421V,L109R/E417R/R427A/Q431D, E112P/S131P/S179V/G204D/E417R/Y421V/R427L,E112P/S131P/S179L/G347D/E417R/Y421V,E112P/R139P/S179V/G204D/I233R/G347D/R427L, E112P/Q159R/E417R/Y421V,E112P/E417R/Y421V, S131P/S179L/I233R/E417R/R427A, R139P/I233R/E417A,Q159R/G347D/E417A/Y421V/Q431D, S179V/E417R/Y421V,I233R/E417R/Y421V/R427L/Q431D, and G347D/E417R, wherein the positionsare numbered with reference to SEQ ID NO: 232. In some furtherembodiments, the polypeptide sequence of the engineeredglycosyltransferase comprises a sequence at least 90% identical to anyof SEQ ID NOS: 348, 350, 352, 354, 356, 358, 360, 362, 364, 366, 368,370, 372, 374 376, 378, 380, 382, 384, 386, 388, 390, 392, 394, 396,398, 400, 402, 404, 406, 408, 410, 412, 414, 416, 418, 420, 422, 424,426, 428, 430, 432, 434, 436, 438, 440, 442, 444, 446, 448, 450, 452,454, 456, 458, 460, 462, 464, 466, 468, 470, 472, 474, 476, 478, 480,482, 484, 486, 488, 490, 492, 494, 496, and/or 498. In some embodiments,the polypeptide sequence of the engineered glycosyltransferase comprisesa sequence at least 95% identical to any of SEQ ID NOS: 348, 350, 352,354, 356, 358, 360, 362, 364, 366, 368, 370, 372, 374, 376, 378, 380,382, 384, 386, 388, 390, 392, 394, 396, 398, 400, 402, 404, 406, 408,410, 412, 414, 416, 418, 420, 422, 424, 426, 428, 430, 432, 434, 436,438, 440, 442, 444, 446, 448, 450, 452, 454, 456, 458, 460, 462, 464,466, 468, 470, 472, 474, 476, 478, 480, 482, 484, 486, 488, 490, 492,494, 496, and/or 498. In some additional embodiments, the polypeptidesequence of the engineered glycosyltransferase comprises any of SEQ IDNOS: 348, 350, 352, 354, 356, 358, 360, 362, 364, 366, 368, 370, 372,374 376, 378, 380, 382, 384, 386, 388, 390, 392, 394, 396, 398, 400,402, 404, 406, 408, 410, 412, 414, 416, 418, 420, 422, 424, 426, 428,430, 432, 434, 436, 438, 440, 442, 444, 446, 448, 450, 452, 454, 456,458, 460, 462, 464, 466, 468, 470, 472, 474, 476, 478, 480, 482, 484,486, 488, 490, 492, 494, 496, and/or 498.

The present invention further provides engineered glycosyltransferases,wherein the polypeptide sequences of the engineered glycosyltransferasescomprise at least one mutation or mutation set at one or more positionsselected from 106/112/204/347/396/417, 106/112/204/347/396/417/427,106/112/204/347/396/417/427/431, 112/204/347/396/417/427,112/204/347/396/417/427/431, and 204/347/396/417/431, wherein thepositions are numbered with reference to SEQ ID NO:348. In someadditional embodiments, the polypeptide sequence of the engineeredglycosyltransferase comprises at least one mutation or mutation set atone or more positions selected from 1065/112P/204D/347D/396Y/417R,106S/112P/204D/347D/396Y/417R/427A,106S/112P/204D/347D/396Y/417R/427A/431D, 112P/204D/347D/396Y/417R/427A,112P/204D/347D/396Y/417R/427A/431D, and 204D/347D/396Y/417R/431D,wherein the positions are numbered with reference to SEQ ID NO:348. Insome embodiments, the polypeptide sequence of the engineeredglycosyltransferase comprises at least one mutation or mutation set atone or more positions selected from L106S/E112P/G204D/G347D/R396Y/E417R,L106S/E112P/G204D/G347D/R396Y/E417R/R427A,L106S/E112P/G204D/G347D/R396Y/E417R/R427A/Q431D,E112P/G204D/G347D/R396Y/E417R/R427A,E112P/G204D/G347D/R396Y/E417R/R427A/Q431D, andG204D/G347D/R396Y/E417R/Q431D, wherein the positions are numbered withreference to SEQ ID NO:348. In some embodiments, the polypeptidesequence of the engineered glycosyltransferase comprises a sequence atleast 90% identical to any of SEQ ID NOS: 500, 502, 504, 506, 508,and/or 510. In some further embodiments, the polypeptide sequence of theengineered glycosyltransferase comprises a sequence at least 95%identical to any of SEQ ID NOS: 500, 502, 504, 506, 508, and/or 510. Insome embodiments, the polypeptide sequence of the engineeredglycosyltransferase comprises SEQ ID NOS: 500, 502, 504, 506, 508,and/or 510.

The present invention also provides engineered glycosyltransferases,wherein the polypeptide sequences of the engineered glycosyltransferasescomprise at least one mutation or mutation set at one or more positionsselected from 14/100, 28/44/365/407, 38/118/290/351/375/401/422,38/178/401, 38/290/351/401/422, 54/413, 74/102/137/161/259/289, 92/118,98/233, 102/161/250/435, 110/222/250/259/435,118/156/178/290/375/401/422, 137/161/435, 137/169,159/169/173/300/424/438, 185/290/401/422, 290/351/401, and 435/438,wherein the positions are numbered with reference to SEQ ID NO:348. Insome embodiments, the polypeptide sequence of the engineeredglycosyltransferase comprises at least one mutation or mutation set atone or more positions selected from 14V/100F, 28M/44V/365I/407E,38R/118A/290E/351G/375P/401L/422M, 38R/178V/401L,38R/290E/351G/401L/422M, 54P/413L, 74W/102K/137G/161L/259S/289S,92L/118A, 98P/233W, 102K/161L/250A/435E, IIOG/222R/250R/259P/435G,118A/156A/178V/290E/375P/401L/422M, 137G/161L/435R, 137G/169G,159M/1695/173G/300Q/424E/438A, 185R/290E/401L/422M, 290E/351G/401L, and435Q/438A, wherein the positions are numbered with reference to SEQ IDNO:348. In some embodiments, the polypeptide sequence of the engineeredglycosyltransferase comprises at least one mutation or mutation set atone or more positions selected from I14V/L100F, I28M/I44V/V365I/A407E,K38R/S118A/D290E/A351G/D375P/W401L/I422M, K38R/A178V/W401L,K38R/D290E/A351G/W401L/I422M, T54P/V413L,R74W/R102K/N137G/D161L/H259S/K289S, I92L/S118A, D98P/I233W,R102K/D161L/T250A/V435E, A110G/K222R/T250R/H259P/V435G,S118A/S156A/A178V/D290E/D375P/W401L/I422M, N137G/D161L/V435R,N137G/D169G, Q159M/D169S/R173G/D300Q/Q424E/M438A,K185R/D290E/W401L/I422M, D290E/A351G/W401L, and V435Q/M438A, wherein thepositions are numbered with reference to SEQ ID NO:348. In someembodiments, the polypeptide sequence of the engineeredglycosyltransferase comprises a sequence at least 90% identical to anyof SEQ ID NOS: 500, 502, 504, 506, 508, 510, 512, 514, 516, 518, 520,222, 524, 526, 528, 530, 532, 534, 536, 538, 540, 542, 544, 546, and/or548. In some embodiments, the polypeptide sequence of the engineeredglycosyltransferase comprises a sequence at least 95% identical to anyof SEQ ID NOS: 500, 502, 504, 506, 508, 510, 512, 514, 516, 518, 520,222, 524, 526, 528, 530, 532, 534, 536, 538, 540, 542, 544, 546, and/or548. In some further embodiments, the polypeptide sequences of theengineered glycosyltransferases comprise any of SEQ ID NOS: 500, 502,504, 506, 508, 510, 512, 514, 516, 518, 520, 222, 524, 526, 528, 530,532, 534, 536, 538, 540, 542, 544, 546, and/or 548.

The present invention also provides engineered glycosyltransferases,wherein the polypeptide sequence of the engineered glycosyltransferasescomprise at least one mutation or mutation set at one or more positionsselected from 14/28/38/74/100/102/118/161/169/178/233/250/407/422/438,14/28/38/74/102/156/159/233/250/289/413/422/424/435/438,14/28/38/100/102/110/159/161/233/259/290/300/351/435,14/28/38/110/137/161/222/289/401,14/28/44/74/98/102/137/159/161/185/222/250, 14/28/44/92,14/28/44/161/169, 14/28/44/375, 14/28/54/161/185/413, 14/28/54/365,14/28/74/98/100/102/161/173/178/233/250/259/290/407,14/28/92/100/102/159/161/169/233, 14/28/92/100/102/159/161/233/351/422,14/28/92/100/102/422/424/435, 14/28/92/100/161/222/233/289/300,14/28/100/137/156/161/222/259/289/365/401/435/438,14/28/100/156/161/250, 14/28/102/118/137/161/185/222/250/259/401,14/28/159/289/290/300, 14/28/159/365/435, 14/28/35,14/38/74/110/156/161/173/178/222/300,14/38/100/102/161/173/178/222/250/375/401/413,44/74/100/102/161/233/365/435/438,14/74/110/159/161/169/173/250/259/290/375/407/422, 14/74/161/375/401,14/92/98/100/159/161/259/365/422/424/435,14/110/156/161/375/401/435/438, 14/159/161/365/435/438,14/161/222/250/259/289/375/401/413, 14/161/222/250/435/438, 14/161/300,28, 28/38/92/98/100/102/156/161, 28/44/74/401, 28/44/92/161/222/300/413,28/44/98/100/102/118, 28/44/118/156/161/222/289/435/M438,28/44/289/290/351/422, 28/44/435/438, 28/54/92/159/161/290,28/54/159/290/438, 28/54/250/439, 28/74/156/159/161/178/300/365/435/438,28/74/156/161/365/407, 28/74/161/290/365,28/92/100/102/110/161/185/250/300/375/435, 28/92/98/100/110/156/161/401,28/92/118/159/222/250/259/300/407, 28/156/161/185/435/438,28/156/161/233/259/300/435, 28/98/100/102/161/185/351/401/435/438,38/161/300/438, 74/98/100/102/110/118/161/178/250/289/290/300/435/438,74/98/100/102/118/156/159/161/435/438, 74/156/161/173/178/424/435,74/375/435, 98/100/118/159/161/300,98/100/156/159/161/178/259/289/290/351/422, 102/137/159/161/422/424,118, 137/159/161/185/300/351/365/435, 156/159/161/169,159/161/222/290/375/407, 159/161/401, and 161/259/289/435/438, whereinthe positions are numbered with reference to SEQ ID NO:548. In someembodiments, the polypeptide sequence of the engineeredglycosyltransferase comprises at least one mutation or mutation set atone or more positions selected from14V/28M/38R/74/100F/102K/118A/161L/169G/178V/233W/250R/407E/422M/438A,14V/28M/38R/74W/102K/156A/159M/233W/250A/289S/413L/422M/424E/435R/438A,14V/28M/38R/100F/102K/110G/159M/161L/233W/259S/290E/300Q/351G/435Q,14V/28M/38R/110G/137G/161L/222R/289S/401L,14V/28M/44V/74W/98P/102K/137G/159M/161L/185R/222R/250A, 14V/28M/44V/92L,14V/28M/44V/161L/169S, 14V/28M/44V/375P, 14V/28M/54P/161L/185R/413L,14V/28M/54P/365I,14V/28M/74W/98P/100F/102K/161L/173G/178V/233W/250A/259S/290E/407E,14V/28M/92L/100F/102K/159M/161L/169S/233W,14V/28M/92L/100F/102K/159M/161L/233W/351G/422M,14V/28M/92L/100F/102K/422M/424E/435Q,14V/28M/92L/100F/161L/222R/233W/289S/300Q,14V/28M/100F/137G/156A/161L/222R/259S/289S/365I/401L/435R/438A,14V/28M/100F/156A/161L/250R,14V/28M/102K/118A/137G/161L/185R/222R/250R/259P/401L,14V/28M/159M/289S/290E/300Q, 14V/28M/159M/365I/435Q, 14V/28M/351G,14V/38R/74W/110G/156A/161L/173G/178V/222R/300Q,14V/38R/100F/102K/161L/173G/178V/222R/250R/375P/401L/413L,44V/74W/100F/102K/161L/I33W/365I/435G/438A,14V/74W/110G/159M/161L/169G/173G/250A/259P/290E/375P/407E/422M,14V/74W/161L/375P/401L,14V/92L/98P/100F/159M/161L/259P/365I/422M/424E/435R,14V/110G/156A/161L/375P/401L/435EM438A, 14V/159M/161L/365I/435E/438A,14V/161L/222R/250R/259S/289S/375P/401L/413L,14V/161L/222R/250R/435Q/438A, 14V/161L/300Q, 28M,28M/38R/92L/98P/100F/102K/156A/161L, 28M/44V/74W/401L,28M/44V/92L/161L/222R/300Q/413L, 28M/44V/98P/100F/102K/118A,28M/44V/118A/156A/161L/222R/289S/435Q/438A, 28M/44V/289S/290E/351G/422M,28M/44V/435R/438A, 28M/54P/92L/159M/161L/290E, 28M/54P/159M/290E/438A,28M/54P/250R/439N 28M/74W/156A/159M/161L/178V/300Q/365I/435Q/438A,28M/74W/156A/161L/365I/407E, 28M/74W/161L/290E/365I,28M/92L/98P/100F/110G/156A/161L/401L,28M/92L/100F/102K/110G/161L/185R/250A/300Q/375P/435Q,28M/92L/118A/159M/222R/250R/259P/300Q/407E,28M/98P/100F/102K/161L/185R/351G/401L/435E/438A,28M/156A/161L/185R/435R/438A, 28M/156A/161L/233W/259S/300Q/435R,38R/161L/300Q/438A,74W/98P/100F/102K/110G/118A/161L/178V/250R/289S/290E/300Q/435E/438A,74W/98P/100F/102K/118A/156A/159M/161L/435E/438A,74W/156A/161L/173G/178V/424E/435E, 74W/375P/435G,98P/100F/118A/159M/161L/300Q,98P/100F/156A/159M/161L/178V/259S/289S/290E/351G/422M,102K/137G/159M/161L/422M/424E, 118A,137G/159M/161L/185R/300Q/351G/365I/435Q, 156A/159M/161L/169S,159M/161L/222R/290E/375P/407E, 159M/161L/401L, and161L/259S/289S/435R/438A, wherein the positions are numbered withreference to SEQ ID NO:548.

In some embodiments, the polypeptide sequence of the engineeredglycosyltransferase comprises at least one mutation or mutation set atone or more positions selected fromI14V/I28M/K38R/R74W/L100F/R102K/S118A/D161L/D169G/A178V/I233W/T250R/A407E/I422M/M438A,I14V/I28M/K38R/R74W/R102K/S156A/Q159M/I233W/T250A/K289S/V413L/I422M/Q424E/V435R/M438A,I14V/I28M/K38R/L100F/R102K/A110G/Q159M/D161L/I233W/H259S/D290E/D300Q/A351G/V435Q,I14V/I28M/K38R/A110G/N137G/D161L/K222R/K289S/W401L,I14V/I28M/I44V/R74W/D98P/R102K/N137G/Q159M/D161L/K185R/K222R/T250A,I14V/I28M/I44V/I92L, I14V/I28M/I44V/D161L/D169S, I14V/I28M/I44V/D375P,I14V/I28M/T54P/D161L/K185R/V413L, I14V/I28M/T54P/V365I,I14V/I28M/R74W/D98P/L100F/R102K/D161L/R173G/A178V/I233W/T250A/H259S/D290E/A407E,I14V/I28M/I92L/L100F/R102K/Q159M/D161L/D169S/I233W,I14V/I28M/I92L/L100F/R102K/Q159M/D161L/I233W/A351G/I422M,I14V/I28M/I92L/L100F/R102K/I422M/Q424E/V435Q,I14V/I28M/I92L/L100F/D161L/K222R/I233W/K289S/D300Q,I14V/I28M/L100F/N137G/S156A/D161L/K222R/H259S/K289S/V365I/W401L/V435R/M438A,I14V/I28M/L100F/S156A/D161L/T250R,I14V/I28M/R102K/S118A/N137G/D161L/K185R/K222R/T250R/H259P/W401L,I14V/I28M/Q159M/K289S/D290E/D300Q, I14V/I28M/Q159M/V365I/V435Q,I14V/I28M/A351G,I14V/K38R/R74W/A110G/S156A/D161L/R173G/A178V/K222R/D300Q,I14V/K38R/L100F/R102K/D161L/R173G/A178V/K222R/T250R/D375P/W401L/V413L,I44V/R74W/L100F/R102K/D161L/I233W/V365I/V435G/M438A,I14V/R74W/A110G/Q159M/D161L/D169G/R173G/T250A/H259P/D290E/D375P/A407E/I422M,I14V/R74W/D161L/D375P/W401L,I14V/I92L/D98P/L100F/Q159M/D161L/H259P/V365I/I422M/Q424E/V435R,I14V/A110G/S156A/D161L/D375P/W401L/V435E/M438A,I14V/Q159M/D161L/V365I/V435E/M438A,I14V/D161L/K222R/T250R/H259S/K289S/D375P/W401L/V413L,I14V/D161L/K222R/T250R/V435Q/M438A, I14V/D161L/D300Q, I28M,I28M/K38R/I92L/D98P/L100F/R102K/S156A/D161L, I28M/I44V/R74W/W401L,I28M/I44V/I92L/D161L/K222R/D300Q/V413L,I28M/I44V/D98P/L100F/R102K/S118A,I28M/I44V/S118A/S156A/D161L/K222R/K289S/V435Q/M438A,I28M/I44V/K289S/D290E/A351G/I422M, I28M/I44V/V435R/M438A,I28M/T54P/I92L/Q159M/D161L/D290E, I28M/T54P/Q159M/D290E/M438A,I28M/T54P/T250R/K439N,I28M/R74W/S156A/Q159M/D161L/A178V/D300Q/V365I/V435Q/M438A,I28M/R74W/S156A/D161L/V365I/A407E, I28M/R74W/D161L/D290E/V365I,I28M/D98P/L100F/R102K/D161L/K185R/A351G/W401L/V435E/M438A,I28M/I92L/L100F/R102K/A10G/D161L/K185R/T250A/D300Q/D375P/V435Q,I28M/I92L/S118A/Q159M/K222R/T250R/H259P/D300Q/A407E,I28M/S156A/D161L/K185R/V435R/M438A,I28M/S156A/D161L/I233W/H259S/D300Q/V435R,I28M/I92L/D98P/L100F/A110G/S156A/D161L/W401L, K38R/D161L/D300Q/M438A,R74W/D98P/L100F/R102K/A110G/S118A/D161L/A178V/T250R/K289S/D290E/D300Q/V435E/M438A, R74W/D98P/L100F/R102K/S118A/S156A/Q159M/D161L/V435E/M438A,R74W/S156A/D161L/R173G/A178V/Q424E/V435E, R74W/D375P/V435G,D98P/L100F/S118A/Q159M/D161L/D300Q,D98P/L100F/S156A/Q159M/D161L/A178V/H259S/K289S/D290E/A351G/I422M,R102K/N137G/Q159M/D161L/I422M/Q424E, S118A,N137G/Q159M/D161L/K185R/D300Q/A351G/V365I/V435Q,S156A/Q159M/D161L/D169S, Q159M/D161L/K222R/D290E/D375P/A407E,Q159M/D161L/W401L, and D161L/H259S/K289S/V435R/M438A, wherein thepositions are numbered with reference to SEQ ID NO:548. In someembodiments, the polypeptide sequence of the engineeredglycosyltransferase comprises a sequence at least 90% identical to anyof SEQ ID NOS: 550, 552, 554, 556, 558, 560, 562, 564, 566, 568, 570,572, 574, 576, 578, 580, 582, 584, 586, 588, 590, 592, 594, 596, 598,600, 602, 604, 606, 608, 610, 612, 614, 616, 618, 620, 622, 624, 626,628, 630, 632, 634, 636, 638, 640, 642, 644, 646, 648, 650, 652, 654,656, 658, 660, 662, 664, 666, 668, 670, 672, 674, 676, 678, and 680,wherein the positions are numbered with reference to SEQ ID NO: 548. Insome embodiments, the polypeptide sequence of the engineeredglycosyltransferase comprises a sequence at least 95% identical to anyof SEQ ID NOS: 550, 552, 554, 556, 558, 560, 562, 564, 566, 568, 570,572, 574, 576, 578, 580, 582, 584, 586, 588, 590, 592, 594, 596, 598,600, 602, 604, 606, 608, 610, 612, 614, 616, 618, 620, 622, 624, 626,628, 630, 632, 634, 636, 638, 640, 642, 644, 646, 648, 650, 652, 654,656, 658, 660, 662, 664, 666, 668, 670, 672, 674, 676, 678, and 680,wherein the positions are numbered with reference to SEQ ID NO: 548.

In some embodiments, the polypeptide sequence of the engineeredglycosyltransferase comprises a sequence any of SEQ ID NOS: 550, 552,554, 556, 558, 560, 562, 564, 566, 568, 570, 572, 574, 576, 578, 580,582, 584, 586, 588, 590, 592, 594, 596, 598, 600, 602, 604, 606, 608,610, 612, 614, 616, 618, 620, 622, 624, 626, 628, 630, 632, 634, 636,638, 640, 642, 644, 646, 648, 650, 652, 654, 656, 658, 660, 662, 664,666, 668, 670, 672, 674, 676, 678, and 680, wherein the positions arenumbered with reference to SEQ ID NO: 548.

The present invention also provides engineered glycosyltransferases,wherein the polypeptide sequences of the engineered glycosyltransferasescomprise at least one mutation or mutation set at one or more positionsselected from 19/44/110/191/198/199/208/300/365,19/75/76/87/92/199/207/208, 19/87/92/191/199/209/413/435,44/76/197/199/208/351, 44/87/92/137/159/199/209,44/87/137/159/169/191/199/208, 44/87/137/191/197/209/289/401,44/87/199/208, 75/76/87/92, 75/76/87/92/290/300, 75/76/87/191,75/76/87/191/197/199/209/300, 75/87/92/169/207/208/300/413/435,75/87/110/137/169/191/199/208/209/289/435,75/87/110/191/197/198/207/208/289/290/300/401/413, 75/87/300,75/110/197/199/208/290/300/401/413, 76/92/199/209,87/92/197/198/199/208/300, 87/137/435, 87/169/191/199/207/209/401/413,87/191/198/199/222/244/289/300/435, 87/92/110/169/199/207/209/290/300,87/92/159/169/191/198/290/413/435, 87/92/159/191/199/208/209/289/290,87/92/208/401, 87/435, 92/137/191/199/209, 92/197/199/207/208/401,137/198/199/207/208/426/435, 137/199/208/209/290/435, 137/365,159/197/199/207/209, 169/191/197/199/207/208,169/197/199/207/209/222/300/413/435, 191/207/208/289/290/413/435, and197/198/199/208/209, wherein the positions are numbered with referenceto SEQ ID NO:562. In some embodiments, the polypeptide sequence of theengineered glycosyltransferase comprises at least one mutation ormutation set at one or more positions selected from19L/44V/110G/191R/198M/199K/208A/300Q/365I,19L/75L/76R/87W/92L/199K/207L/208A,19L/87W/92L/191R/199K/209G/413L/435Q, 44V/76R/197R/199K/208D/351G,44V/87W/92L/137G/159M/199K/209G, 44V/87W/137G/159M/1695/191R/199K/208D,44V/87W/137G/191R/197R/209G/289S/401L, 44V/87W/199K/208A,75L/76R/87W/92L, 75L/76R/87W/92L/290E/300Q, 75L/76R/87W/191R,75L/76R/87W/191R/197R/199K/209G/300Q,75L/87W/92L/169S/207L/208A/300Q/413L/435Q,75L/87W/110G/137G/1695/191R/199K/208A/209G/289S/435Q,75L/87W/110G/191R/197R/198M/207L/208D/289S/290E/300Q/401L/413L,75L/87W/300Q, 75L/110G/197R/199K/208A/290E/300Q/401L/413L,76R/92L/199K/209G, 87L/92L/197R/198M/199K/208D/300Q, 87W/137G/435R,87W/169S/191R/199K/207L/209G/401L/413L,87W/191R/198M/199K/222R/244L/289S/300Q/435R,87W/92L/110G/169S/199K/207L/209G/290E/300Q,87W/92L/159M/1695/191R/198M/290E/413L/435Q,87W/92L/159M/191R/199K/208A/209G/289S/290E, 87W/92L/208A/401L, 87W/435R,92L/137G/191R/199K/209G, 92L/197R/199K/207L/208D/401L,137G/198M/199K/207L/208D/426V/435R, 137G/199K/208A/209G/290E/435R,137G/365I, 159M/197R/199K/207L/209G, 169S/191R/197R/199K/207L/208D,169S/197R/199K/207L/209G/222R/300Q/413L/435R,191R/207L/208A/289S/290E/413L/435Q, and 197R/198M/199K/208D/209G,wherein the positions are numbered with reference to SEQ ID NO:562. Insome embodiments, the polypeptide sequence of the engineeredglycosyltransferase comprises at least one mutation or mutation set atone or more positions selected fromV19L/I44V/A110G/S191R/I198M/A199K/K208A/D300Q/V365I,V19L/I75L/S76R/M87W/I92L/A199K/I207L/K208A,V19L/M87W/I92L/S191R/A199K/Q209G/V413L/V435Q,I44V/S76R/Q197R/A199K/K208D/A351G,I44V/M87W/I92L/N137G/Q159M/A199K/Q209G,I44V/M87W/N137G/Q159M/G169S/S191R/A199K/K208D,I44V/M87W/N137G/S191R/Q197R/Q209G/K289S/W401L, I44V/M87W/A199K/K208A,I75L/S76R/M87W/I92L, I75L/S76R/M87W/I92L/D290E/D300Q,I75L/S76R/M87W/S191R, I75L/S76R/M87W/S191R/Q197R/A199K/Q209G/D300Q,I75L/M87W/I92L/G169S/I207L/K208A/D300Q/V413L/V435Q,I75L/M87W/A110G/N137G/G169S/S191R/A199K/K208A/Q209G/K289S/V435Q,I75L/M87W/A110G/S191R/Q197R/I198M/I207L/K208D/K289S/D290E/D300Q/W401L/V41L, I75L/M87W/D300Q,I75L/A110G/Q197R/A199K/K208A/D290E/D300Q/W401L/V413L,S76R/I92L/A199K/Q209G, M87L/I92L/Q197R/I198M/A199K/K208D/D300Q,M87W/N137G/V435R, M87W/G169S/S191R/A199K/I207L/Q209G/W401L/V413L,M87W/S191R/I198M/A199K/K222R/P244L/K289S/D300Q/V435R,M87W/I92L/A110G/G169S/A199K/I207L/Q209G/D290E/D300Q,M87W/I92L/Q159M/G169S/S191R/I198M/D290E/V413L/V435Q,M87W/I92L/Q159M/S191R/A199K/K208A/Q209G/K289S/D290E,M87W/I92L/K208A/W401L, M87W/V435R, I92L/N137G/S191R/A199K/Q209G,I92L/Q197R/A199K/I207L/K208D/W401L,N137G/I198M/A199K/I207L/K208D/A426V/V435R,N137G/A199K/K208A/Q209G/D290E/V435R, N137G/V365I,Q159M/Q197R/A199K/I207L/Q209G, G169S/S191R/Q197R/A199K/I207L/K208D,G169S/Q197R/A199K/I207L/Q209G/K222R/D300Q/V413L/V435R,S191R/I207L/K208A/K289S/D290E/V413L/V435Q, andQ197R/I198M/A199K/K208D/Q209G, wherein the positions are numbered withreference to SEQ ID NO:562. In some embodiments, the polypeptidesequence of the engineered glycosyltransferase comprises a sequence atleast 90% identical to any of SEQ ID NOS: 682, 684, 686, 688, 690, 692,694, 696, 698, 700, 702, 704, 706, 708, 710, 712, 714, 716, 718, 720,722, 724, 726, 728, 730, 732, 734, 736, 738, 740, 742, 744, 746, 748,750, 752, and/or 754. In some further embodiments, the polypeptidesequence of the engineered glycosyltransferase comprises a sequence atleast 95% identical to any of SEQ ID NOS: 682, 684, 686, 688, 690, 692,694, 696, 698, 700, 702, 704, 706, 708, 710, 712, 714, 716, 718, 720,722, 724, 726, 728, 730, 732, 734, 736, 738, 740, 742, 744, 746, 748,750, 752, and/or 754. In some embodiments, the polypeptide sequence ofthe engineered glycosyltransferase comprises SEQ ID NOS: 682, 684, 686,688, 690, 692, 694, 696, 698, 700, 702, 704, 706, 708, 710, 712, 714,716, 718, 720, 722, 724, 726, 728, 730, 732, 734, 736, 738, 740, 742,744, 746, 748, 750, 752, and/or 754.

The present invention also provides engineered glycosyltransferases,wherein the polypeptide sequences of the engineered glycosyltransferasescomprise at least one mutation or mutation set at one or more positionsselected from 22, 25, 51, 56, 71, 78, 80, 81, 88, 157,185/208/230/252/255/290/365, 189/206/208/365, 200, 208/365/435, 243,245, 249, 259, 262/401, 279, 282, 284, 304/322/365/401, 308, 338, 339,352, 362, 364, 365/401/413/435, 366, and 374, wherein the positions arenumbered with reference to SEQ ID NO:696. In some embodiments, thepolypeptide sequence of the engineered glycosyltransferase comprises atleast one mutation or mutation set at one or more positions selectedfrom 22L, 25L, 25V, 51A, 56L, 71R, 78E, 78F, 78G, 78I, 78K, 78M, 78P,78Q, 78R, 80L, 81C, 88I, 88K, 88V, 157G, 157Q,185R/208A/230S/252N/255N/290E/365I, 189L/206K/208A/365I, 200N, 200S,208A/365I/435Q, 243C, 243L, 243M, 243V, 243Y, 245G, 249E, 249H, 249I,249M, 249N, 249P, 2495, 249T, 249Y, 259G, 259, 259Y, 262S/401L, 279G,282T, 284T, 304P/322S/365I/401L, 308F, 308Y, 338C, 339D, 352Q, 362T,364G, 365I/401L/413L/435Q, 366A, and 374T, wherein the positions arenumbered with reference to SEQ ID NO:696. In some additionalembodiments, the polypeptide sequence of the engineeredglycosyltransferase comprises at least one mutation or mutation set atone or more positions selected from P22L, I25L, I25V, K51A, N56L, Q71R,L78E, L78F, L78G, L78I, L78K, L78M, L78P, L78Q, L78R, T80L, H81C, R88I,R88K, R88V, L157G, L157Q, K185R/K208A/E230S/S252N/S255N/D290E/V365I,I189L/M206K/K208A/V365I, K200N, K200S, K208A/V365I/V435Q, I243C, I243L,I243M, I243V, I243Y, L245G, L249E, L249H, L249I, L249M, L249N, L249P,L249S, L249T, L249Y, H259G, H259S, H259Y, T262S/W401L, S279G, S282T,S284T, S304P/P322S/V365I/W401L, R308F, R308Y, V338C, P339D, F352Q,L362T, S364G, V365I/W401L/V413L/V435Q, C366A, and S374T, wherein thepositions are numbered with reference to SEQ ID NO:696. In someembodiments, the polypeptide sequence of the engineeredglycosyltransferase comprises a sequence at least 90% identical to anyof SEQ ID NOS: 4684, 4686, 4688, 4690, 4692, 4694, 4696, 4698, 4700,4702, 4704, 4706, 4708, 4710, 4712, 4714, 4716, 4718, 4720, 4722, 4724,4726, 4728, 4730, 4732, 4734, 4736, 4738, 4740, 4742, 4744, 4746, 4748,4750, 4752, 4754, 4756, 4758, 4760, 4762, 4764, 4766, 4768, 4770, 4772,4774, 4776, 4778, 4780, 4782, 4784, 4786, 4788, 4790, 4792, 4794, 4796,4798, 4800, 4902, 4804, 4806, 4808, 4810, and 4812. In some embodiments,the polypeptide sequence of the engineered glycosyltransferase comprisesa sequence at least 95% identical to any of SEQ ID NOS: 4684, 4686,4688, 4690, 4692, 4694, 4696, 4698, 4700, 4702, 4704, 4706, 4708, 4710,4712, 4714, 4716, 4718, 4720, 4722, 4724, 4726, 4728, 4730, 4732, 4734,4736, 4738, 4740, 4742, 4744, 4746, 4748, 4750, 4752, 4754, 4756, 4758,4760, 4762, 4764, 4766, 4768, 4770, 4772, 4774, 4776, 4778, 4780, 4782,4784, 4786, 4788, 4790, 4792, 4794, 4796, 4798, 4800, 4902, 4804, 4806,4808, 4810, and 4812. In some embodiments, the polypeptide sequence ofthe engineered glycosyltransferase comprises any of SEQ ID NOS: 4684,4686, 4688, 4690, 4692, 4694, 4696, 4698, 4700, 4702, 4704, 4706, 4708,4710, 4712, 4714, 4716, 4718, 4720, 4722, 4724, 4726, 4728, 4730, 4732,4734, 4736, 4738, 4740, 4742, 4744, 4746, 4748, 4750, 4752, 4754, 4756,4758, 4760, 4762, 4764, 4766, 4768, 4770, 4772, 4774, 4776, 4778, 4780,4782, 4784, 4786, 4788, 4790, 4792, 4794, 4796, 4798, 4800, 4902, 4804,4806, 4808, 4810, and 4812.

The present invention also provides engineered glycosyltransferases,wherein the polypeptide sequences of the engineered glycosyltransferasescomprise at least one mutation or mutation set at one or more positionsselected from 51/56, 51/56/243/249/282/353/362/366,51/56/243/249/308/362/364, 51/56/249/353, 51/56/249/362/364,51/56/249/362/366, 51/243/249/308/353, 51/243/249/348/362/366, 51/249,51/249/282/284/364, 51/249/282/353/366, 51/249/284/308/362/366,51/249/353/362/364, 51/353/362, 56, 56/243/249/282/364/366,56/243/364/366, 56/249, 56/249/284/353, 56/249/353, 56/284/366,243/249/282/284/362/364/366, 243/249/308/353/366, 243/249/353/362/366,243/282/353/362/364,243/282/362/364/366, 243/308/353,249/353/362/366,282, 308/366, and 362/366, wherein the positions are numbered withreference to SEQ ID NO: 4684.

In some embodiments, the polypeptide sequences of the engineeredglycosyltransferases comprise at least one mutation or mutation set atone or more positions selected from 51A/56H,51A/56H/243L/249E/282T/353Y/362M/366S, 51A/56H/243L/249E/308F/362M/364G,51A/56H/249M/362M/364G, 51A/56H/249M/362M/366S, 51A/56H/249Y/353Y,51A/243L/249E/308F/353Y, 51A/243L/249E/348S/362M/366V,51A/249E/353Y/362M/364G, 51A/249M, 51A/249M/282T/284T/364G,51A/249M/282T/353Y/366S, 51A/249Y/284T/308F/362M/366V, 51A/353Y/362M,56H, 56H/243L/249E/282T/364G/366V, 56H/243L/364G/366V,56H/249M/284T/353Y, 56H/249M/353Y, 56H/249Y, 56H/284T/366V,243L/249E/282T/284T/362M/364G/366S, 243L/249M/308F/353Y/366A,243L/249Y/353Y/362M/366S, 243L/282T/353Y/362M/364G,243L/282T/362M/364G/366V, 243L/308F/353Y, 249Y/353Y/362M/366S, 282T,308F/366A, and 362M/366A, wherein the positions are numbered withreference to SEQ ID NO: 4684. In some embodiments, the polypeptidesequences of the engineered glycosyltransferases comprise at least onemutation or mutation set at one or more positions selected fromK51A/N56H, K51A/N56H/I243L/L249E/S282T/W353Y/L362M/C366S,K51A/N56H/I243L/L249E/R308F/L362M/S364G, K51A/N56H/L249M/L362M/S364G,K51A/N56H/L249M/L362M/C366S, K51A/N56H/L249Y/W353Y,K51A/I243L/L249E/R308F/W353Y, K51A/I243L/L249E/A348S/L362M/C366V,K51A/L249E/W353Y/L362M/S364G, K51A/L249M, K51A/L249M/S282T/S284T/S364G,K51A/L249M/S282T/W353Y/C366S, K51A/L249Y/S284T/R308F/L362M/C366V,K51A/W353Y/L362M, N56H, N56H/I243L/L249E/S282T/S364G/C366V,N56H/I243L/S364G/C366V, N56H/L249M/S284T/W353Y, N56H/L249M/W353Y,N56H/L249Y, N56H/S284T/C366V, I243L/L249E/S282T/S284T/L362M/S364G/C366S,I243L/L249M/R308F/W353Y/C366A, I243L/L249Y/W353Y/L362M/C366S,I243L/S282T/W353Y/L362M/S364G, I243L/S282T/L362M/S364G/C366V,I243L/R308F/W353Y, L249Y/W353Y/L362M/C366S, S282T, R308F/C366A, andL362M/C366A, wherein the positions are numbered with reference to SEQ IDNO: 4684. In some embodiments, the polypeptide sequence of theengineered glycosyltransferase comprises a sequence at least 90%identical to any of SEQ ID NOS: 4814, 4816, 4818, 4820, 4822, 4824,4826, 4828, 4830, 4832, 4834, 4836, 4838, 4840, 4842, 4844, 4846, 4848,4850, 4852, 4854, 4856, 4858, 4860, 4862, 4864, 4866, 4868, 4870, 4872,and 4874. In some additional embodiments, the polypeptide sequence ofthe engineered glycosyltransferase comprises a sequence at least 95%identical to any of SEQ ID NOS: 4814, 4816, 4818, 4820, 4822, 4824,4826, 4828, 4830, 4832, 4834, 4836, 4838, 4840, 4842, 4844, 4846, 4848,4850, 4852, 4854, 4856, 4858, 4860, 4862, 4864, 4866, 4868, 4870, 4872,and 4874. In some further embodiments, the polypeptide sequence of theengineered glycosyltransferase comprises any of SEQ ID NOS: 4814, 4816,4818, 4820, 4822, 4824, 4826, 4828, 4830, 4832, 4834, 4836, 4838, 4840,4842, 4844, 4846, 4848, 4850, 4852, 4854, 4856, 4858, 4860, 4862, 4864,4866, 4868, 4870, 4872, and 4874.

The present invention also provides engineered glycosyltransferases,wherein the polypeptide sequences of the engineered glycosyltransferasescomprise at least one mutation or mutation set at one or more positionsselected from 25, 25/56/353, 25/243/249/259/366, 25/243/249/362,25/362/366, 51, 51/56/200/243/249/259/338, 51/56/362,51/71/249/279/284/362/366, 56/243/249, 56/362/366, 70/198/259/313, 85,88, 88/173, 110, 159, 163, 171, 174, 175, 177, 198, 198/313,198/313/428, 200/243/249/259, 208, 208/320, 209, 209/234, 222, 226, 234,234/408, 243/338/362/366, 253, 256, 259, 265, 272, 289, 322, 336,353/362, 405, 411, 428, and 439, wherein the positions are numbered withreference to SEQ ID NO: 4838. In some embodiments, the polypeptidesequences of the engineered glycosyltransferases comprise at least onemutation or mutation set at one or more positions selected from 25L,25L/56L/353Y, 25V/243M/249I/362M, 25V/243M/249Y/259G/366A,25V/362M/366S, 51A, 51A/56L/200S/243M/249I/259G/338C, 51A/56L/362M,51A/71R/249M/279G/284T/362M/366S, 56L/243M/249M, 56L/243M/249Y,56L/362M/366A, 70S/198D/259E/313S, 85E, 88C/173S, 88I, 110S, 159N, 163K,171D, 171E, 171P, 171V, 174E, 174S, 175N, 177K, 177P, 177S, 198D,198D/313S, 198D/313S/428S, 198E, 198S, 198T, 200S/243M/249Y/259G, 208E,208G, 208I, 208L, 208N, 208T, 208V, 208W/320I, 209A, 209E/234Q, 222P,226R, 226T, 234A, 234E, 234H/408D, 234T, 243M/338C/362M/366S, 253A,256A, 259N, 259S, 259T, 265A, 272D, 289H, 289T, 322V, 336E, 353Y/362M,405Q, 411T, 428K, 439D, and 439Q, wherein the positions are numberedwith reference to SEQ ID NO: 4838. In some additional embodiments, thepolypeptide sequences of the engineered glycosyltransferases comprise atleast one mutation or mutation set at one or more positions selectedfrom I25L, I25L/H56L/W353Y, I25V/L243M/E249I/L362M,I25V/L243M/E249Y/H259G/V366A, I25V/L362M/V366S, K51A,K51A/H56L/K200S/L243M/E249I/H259G/V338C, K51A/H56L/L362M,K51A/Q71R/E249M/S279G/S284T/L362M/V366S, H56L/L243M/E249M,H56L/L243M/E249Y, H56L/L362M/V366A, P70S/I198D/H259E/F313S, A85E,R88C/R173S, R88I, A110S, Q159N, L163K, K171D, K171E, K171P, K171V,L174E, L174S, E175N, Q177K, Q177P, Q177S, I198D, I198D/F313S,I198D/F313S/V428S, I198E, I198S, I198T, K200S/L243M/E249Y/H259G, K208E,K208G, K208I, K208L, K208N, K208T, K208V, K208W/V320I, Q209A,Q209E/R234Q, K222P, E226R, E226T, R234A, R234E, R234H/N408D, R234T,L243M/V338C/L362M/V366S, S253A, L256A, H259N, H259S, H259T, Q265A,P272D, K289H, K289T, P322V, K336E, W353Y/L362M, E405Q, R411T, V428K,K439D, and K439Q, wherein the positions are numbered with reference toSEQ ID NO: 4838. In some embodiments, the polypeptide sequence of theengineered glycosyltransferase comprises a sequence at least 90%identical to any of SEQ ID NOS: 4876, 4878, 4880, 4882, 4884, 4886,4888, 4890, 4892, 4894, 4896, 4898, 4900, 4902, 4904, 4906, 4908, 4910,4912, 4914, 4916, 4918, 4920, 4922, 4924, 4926, 4928, 4930, 4932, 4934,4936, 4938, 4940, 4942, 4944, 4946, 4948, 4950, 4952, 4954, 4956, 4958,4960, 4962, 4964, 4966, 4968, 4970, 4972, 4974, 4976, 4978, 4980, 4982,4984, 4986, 4988, 4990, 4992, 4994, 4996, 4998, 5000, 5002, 5004, 5006,5008, 5010, 5012, 5014, and 5016. In some embodiments, the polypeptidesequence of the engineered glycosyltransferase comprises a sequence atleast 95% identical to any of SEQ ID NOS: 4876, 4878, 4880, 4882, 4884,4886, 4888, 4890, 4892, 4894, 4896, 4898, 4900, 4902, 4904, 4906, 4908,4910, 4912, 4914, 4916, 4918, 4920, 4922, 4924, 4926, 4928, 4930, 4932,4934, 4936, 4938, 4940, 4942, 4944, 4946, 4948, 4950, 4952, 4954, 4956,4958, 4960, 4962, 4964, 4966, 4968, 4970, 4972, 4974, 4976, 4978, 4980,4982, 4984, 4986, 4988, 4990, 4992, 4994, 4996, 4998, 5000, 5002, 5004,5006, 5008, 5010, 5012, 5014, and 5016. In some embodiments, thepolypeptide sequence of the engineered glycosyltransferase comprises anyof SEQ ID NOS: 4876, 4878, 4880, 4882, 4884, 4886, 4888, 4890, 4892,4894, 4896, 4898, 4900, 4902, 4904, 4906, 4908, 4910, 4912, 4914, 4916,4918, 4920, 4922, 4924, 4926, 4928, 4930, 4932, 4934, 4936, 4938, 4940,4942, 4944, 4946, 4948, 4950, 4952, 4954, 4956, 4958, 4960, 4962, 4964,4966, 4968, 4970, 4972, 4974, 4976, 4978, 4980, 4982, 4984, 4986, 4988,4990, 4992, 4994, 4996, 4998, 5000, 5002, 5004, 5006, 5008, 5010, 5012,5014, and 5016.

The present invention also provides engineered glycosyltransferases,wherein the polypeptide sequences of the engineered glycosyltransferasescomprise at least one mutation or mutation set at one or more positionsselected from 8, 9, 25, 25/198/209, 25/200/209/338, 25/200/243/249,25/209/243, 25/209/243/249, 25/209/243/259, 25/209/249/259/366,25/209/259, 25/209/279/366, 25/209/289/366, 25/249, 25/259/279,25/259/279/289, 25/279/284, 25/279/284/289, 25/289, 34, 53, 54, 55, 61,69, 70, 73, 79, 87, 91, 107, 108, 111, 141, 153, 158, 174, 190, 194,198, 198/200/209/243/249/289, 198/249/338, 200/209/366,201, 205, 209,209/249/259/279/338/366, 209/249/259/289, 209/249/279/284/289,209/249/338, 209/279/289, 209/366, 234/297, 238, 247, 249/259, 252, 253,254, 256, 259/366, 279, 279/338, 289, 297, 308, 321, 322, 327, 336, 338,341, 342, 364, 366, 388, 392, 411, 412, 414, 426, 430, 432, 446, and449, wherein the positions are numbered with reference to SEQ IDNO:4876. In some embodiments, the polypeptide sequence of the engineeredglycosyltransferase comprises at least one mutation or mutation set atone or more positions selected from 8D, 8L, 9G, 25H/209E/243M/249Y, 25L,25L/198D/209E, 25L/200S/209E/338C, 25L/200S/243M/249M, 25L/209E/243M,25L/209E/243M/249Y, 25L/209E/243M/259G, 25L/209E/249I/259G/366A,25L/209E/259G, 25L/209E/279G/366A, 25L/209E/289T/366A, 25L/249I,25L/259G/279G, 25L/259G/279G/289T, 25L/279G/284T, 25L/279G/284T/289T,25L/289T, 34I, 53C, 53L, 53R, 53V, 54P, 54V, 55T, 55W, 615, 69N, 69Q,69S, 69T, 70K, 73S, 79G, 79S, 79V, 87L, 87M, 87R, 91Q, 91R, 91T, 107A,107C, 107T, 108C, 111C, 111G, 141M, 1535, 158Q, 174M, 190R, 194Q, 198D,198D/200S/209E/243M/249Y/289T, 198D/249M/338C, 200S/209E/366A, 201P,201T, 205P, 209E, 209E/249I/259G/289T, 209E/249M/338C,209E/249Y/259G/279G/338C/366A, 209E/249Y/279G/284T/289T, 209E/279G/289T,209E/366A, 234H/297A, 238K, 247A, 247L, 249M/259G, 252A, 252E, 252Q,253G, 253P, 254G, 254M, 254P, 256D, 256T, 256W, 259G/366A, 279G,279G/338C, 289T, 297A, 308C, 308L, 308T, 321D, 322M, 322R, 322T, 327L,336M, 336Y, 338C, 341V, 342R, 364S, 366A, 388T, 388V, 392H, 392Q, 411E,411S, 412H, 412T, 414L, 426T, 430R, 432T, 446F, and 449L, wherein thepositions are numbered with reference to SEQ ID NO:4876. In someadditional embodiments, the polypeptide sequence of the engineeredglycosyltransferase comprises at least one mutation or mutation set atone or more positions selected from T8D, T8L, V9G,I25H/Q209E/L243M/E249Y, I25L, I25L/I198D/Q209E, I25L/K200S/Q209E/V338C,I25L/K200S/L243M/E249M, I25L/Q209E/L243M, I25L/Q209E/L243M/E249Y,I25L/Q209E/L243M/H259G, I25L/Q209E/E249I/H259G/V366A, I25L/Q209E/H259G,I25L/Q209E/S279G/V366A, I25L/Q209E/K289T/V366A, I25L/E249I,I25L/H259G/S279G, I25L/H259G/S279G/K289T, I25L/S279G/S284T,I25L/S279G/S284T/K289T, I25L/K289T, V34I, K53C, K53L, K53R, K53V, T54P,T54V, S55T, S55W, T61S, D69N, D69Q, D69S, D69T, P70K, E73S, P79G, P79S,P79V, W87L, W87M, W87R, I91Q, I91R, I91T, L107A, L107C, L107T, M108C,SIIC, S111G, L141M, A153S, P158Q, L174M, K190R, S194Q, I198D,I198D/K200S/Q209E/L243M/E249Y/K289T, I198D/E249M/V338C,K200S/Q209E/V366A, E201P, E201T, K205P, Q209E, Q209E/E249I/H259G/K289T,Q209E/E249M/V338C, Q209E/E249Y/H259G/S279G/V338C/V366A,Q209E/E249Y/S279G/S284T/K289T, Q209E/S279G/K289T, Q209E/V366A,R234H/G297A, A238K, K247A, K247L, E249M/H259G, S252A, S252E, S252Q,S253G, S253P, S254G, S254M, S254P, L256D, L256T, L256W, H259G/V366A,S279G, S279G/V338C, K289T, G297A, R308C, R308L, R308T, E321D, P322M,P322R, P322T, F327L, K336M, K336Y, V338C, Q341V, E342R, G364S, V366A,S388T, S388V, K392H, K392Q, R411E, R411S, R412H, R412T, M414L, A426T,K430R, K432T, S446F, and S449L, wherein the positions are numbered withreference to SEQ ID NO:4876. In some embodiments, the polypeptidesequence of the engineered glycosyltransferase comprises a sequence atleast 90% identical to any of SEQ ID NOS: 5018, 5020, 5022, 5024, 5026,5028, 5030, 5032, 5034, 5036, 5038, 5040, 5042, 5044, 5046, 5048, 5050,5052, 5054, 5056, 5058, 5060, 5062, 5064, 5066, 5068, 5070, 5072, 5074,5076, 5078, 5080, 5082, 5084, 5086, 5088, 5090, 5092, 5094, 5096, 5098,5100, 5102, 5104, 5106, 5108, 5110, 5112, 5114, 5116, 5118, 5120, 5122,5124, 5126, 5128, 5130, 5132, 5134, 5136, 5138, 5140, 5142, 5144, 5146,5148, 5150, 5152, 5154, 5156, 5158, 5160, 5162, 5164, 5166, 5168, 5170,5172, 5174, 5176, 5178, 5180, 5182, 5184, 5186, 5188, 5190, 5192, 5194,5196, 5198, 5200, 5202, 5204, 5206, 5208, 5210, 5212, 5214, 5216, 5218,5220, 5222, 5224, 5226, 5228, 5230, 5232, 5234, 5236, 5238, 5240, 5242,5244, 5246, 5248, 5250, 5252, 5254, 5256, 5258, and 5260. In someembodiments, the polypeptide sequence of the engineeredglycosyltransferase comprises a sequence at least 95% identical to anyof SEQ ID NOS: 5018, 5020, 5022, 5024, 5026, 5028, 5030, 5032, 5034,5036, 5038, 5040, 5042, 5044, 5046, 5048, 5050, 5052, 5054, 5056, 5058,5060, 5062, 5064, 5066, 5068, 5070, 5072, 5074, 5076, 5078, 5080, 5082,5084, 5086, 5088, 5090, 5092, 5094, 5096, 5098, 5100, 5102, 5104, 5106,5108, 5110, 5112, 5114, 5116, 5118, 5120, 5122, 5124, 5126, 5128, 5130,5132, 5134, 5136, 5138, 5140, 5142, 5144, 5146, 5148, 5150, 5152, 5154,5156, 5158, 5160, 5162, 5164, 5166, 5168, 5170, 5172, 5174, 5176, 5178,5180, 5182, 5184, 5186, 5188, 5190, 5192, 5194, 5196, 5198, 5200, 5202,5204, 5206, 5208, 5210, 5212, 5214, 5216, 5218, 5220, 5222, 5224, 5226,5228, 5230, 5232, 5234, 5236, 5238, 5240, 5242, 5244, 5246, 5248, 5250,5252, 5254, 5256, 5258, and 5260. In some embodiments, the polypeptidesequence of the engineered glycosyltransferase comprises any of SEQ IDNOS: 5018, 5020, 5022, 5024, 5026, 5028, 5030, 5032, 5034, 5036, 5038,5040, 5042, 5044, 5046, 5048, 5050, 5052, 5054, 5056, 5058, 5060, 5062,5064, 5066, 5068, 5070, 5072, 5074, 5076, 5078, 5080, 5082, 5084, 5086,5088, 5090, 5092, 5094, 5096, 5098, 5100, 5102, 5104, 5106, 5108, 5110,5112, 5114, 5116, 5118, 5120, 5122, 5124, 5126, 5128, 5130, 5132, 5134,5136, 5138, 5140, 5142, 5144, 5146, 5148, 5150, 5152, 5154, 5156, 5158,5160, 5162, 5164, 5166, 5168, 5170, 5172, 5174, 5176, 5178, 5180, 5182,5184, 5186, 5188, 5190, 5192, 5194, 5196, 5198, 5200, 5202, 5204, 5206,5208, 5210, 5212, 5214, 5216, 5218, 5220, 5222, 5224, 5226, 5228, 5230,5232, 5234, 5236, 5238, 5240, 5242, 5244, 5246, 5248, 5250, 5252, 5254,5256, 5258, and 5260.

The present invention also provides engineered glycosyltransferases,wherein the polypeptide sequences of the engineered glycosyltransferasescomprise at least one mutation or mutation set at one or more positionsselected from 88, 88/110, 88/110/159/198/200,88/110/159/198/234/259/265, 88/110/159/198/259, 88/110/198, 88/110/234,88/159/198, 88/198, 88/198/200/259, 88/198/259, 88/259, 88/259/265,110/259/265/411, 159/198/411, 159/259/411,171/174/175/177/208/320/428/439, 171/175/177/208/320/428, 171/177,171/177/226/428/439, 171/208/320, 171/208/320/428, 171/208/428,174/175/177/208/320/428, 174/175/428, 174/320, 198, 198/200,198/200/234, 198/234, 198/259, 208, 208/320/331/428, 208/320/428,208/428, 234, 259, 320/428, and 428, wherein the positions are numberedwith reference to SEQ ID NO: 5066. In some embodiments, the polypeptidesequences of the engineered glycosyltransferases comprise at least onemutation or mutation set at one or more positions selected from 88,88I/110D, 88I/110D/159N/198D/234T/259N/265A, 88I/110D/159N/198D/259E,88I/110D/159N/198E/200S, 88I/110D/198D, 88I/110D/198S, 88I/110D/234T,88I/159N/198D, 88I/198D/200S/259E, 88I/198D/259E, 88I/198S,88I/259E/265A, 88I/259N, 110D/259N/265A/411T, 159N/198E/411T,159N/259E/411T, 171E/177P/226T/428K/439D, 171E/208E/320I,171E/208E/320I/428K, 171E/208E/428K,171P/174E/175N/177P/208W/320I/428K/439D, 171P/175N/177P/208L/320I/428K,171P/177P, 174E/175N/177P/208E/320I/428K, 174E/175N/428K, 174E/320I,198D, 198D/200S/234E, 198D/234Q, 198E/200S, 198E/259E, 208E,208E/320I/428K, 208E/428K, 208L/320I/331C/428K, 234E, 259E, 320I/428K,and 428K, wherein the positions are numbered with reference to SEQ IDNO: 5066. In some embodiments, the polypeptide sequences of theengineered glycosyltransferases comprise at least one mutation ormutation set at one or more positions selected from R88I, R88I/A101D,R88I/A110D/Q159N/I198D/R234T/G259N/Q265A, R88I/A110D/Q159N/I198D/G259E,R88I/A110D/Q159N/I198E/K200S, R88I/A110D/I198D, R88I/A110D/I198S,R88I/A110D/R234T, R88I/Q159N/I198D, R88I/I198D/K200S/G259E,R88I/I198D/G259E, R88I/I198S, R88I/G259E/Q265A, R88I/G259N,A110D/G259N/Q265A/R411T, Q159N/I198E/R411T, Q159N/G259E/R411T,K171E/Q177P/E226T/V428K/K439D, K171E/K208E/V320I,K171E/K208E/V320I/V428K, K171E/K208E/V428K,K171P/L174E/E175N/Q177P/K208W/V320I/V428K/K439D,K171P/E175N/Q177P/K208L/V320I/V428K, K171P/Q177P,L174E/E175N/Q177P/K208E/V320I/V428K, L174E/E175N/V428K, L174E/V320I,I198D, I198D/K200S/R234E, I198D/R234Q, I198E/K200S, I98E/G259E, K208E,K208E/V320I/V428K, K208E/V428K, K208L/V320I/R331C/V428K, R234E, G259E,V320I/V428K, and V428K, wherein the positions are numbered withreference to SEQ ID NO: 5066. In some embodiments, the polypeptidesequence of the engineered glycosyltransferase comprises a sequence atleast 90% identical to any of SEQ ID NOS: 5262, 5264, 5266, 5268, 5270,5272, 5274, 5276, 5278, 5280, 5282, 5284, 5286, 5288, 5290, 5292, 5294,5296, 5298, 5300, 5302, 5304, 5306, 5308, 5310, 5312, 5314, 5316, 5318,5320, 5322, 5324, 5326, 5328, 5330, 5332, 5334, 5336, 5338, 5340, and5342. In some embodiments, the polypeptide sequence of the engineeredglycosyltransferase comprises a sequence at least 95% identical to anyof SEQ ID NOS: 5262, 5264, 5266, 5268, 5270, 5272, 5274, 5276, 5278,5280, 5282, 5284, 5286, 5288, 5290, 5292, 5294, 5296, 5298, 5300, 5302,5304, 5306, 5308, 5310, 5312, 5314, 5316, 5318, 5320, 5322, 5324, 5326,5328, 5330, 5332, 5334, 5336, 5338, 5340, and 5342. In some embodiments,the polypeptide sequence of the engineered glycosyltransferase comprisesany of SEQ ID NOS: 5262, 5264, 5266, 5268, 5270, 5272, 5274, 5276, 5278,5280, 5282, 5284, 5286, 5288, 5290, 5292, 5294, 5296, 5298, 5300, 5302,5304, 5306, 5308, 5310, 5312, 5314, 5316, 5318, 5320, 5322, 5324, 5326,5328, 5330, 5332, 5334, 5336, 5338, 5340, and 5342.

The present invention also provides engineered glycosyltransferases,wherein the polypeptide sequences of the engineered glycosyltransferasescomprise at least one mutation or mutation set at one or more positionsselected from 2, 7, 25/61/208/252/253, 25/61/428, 25/107/208/320/428,25/208/247/252/253/364/428, 25/247/249/252/364/428, 38,53/54/55/153/201, 53/54/308/392, 53/171/308/392, 54,61/107/208/252/253/254/364/428, 61/208/252/254/428, 64, 68, 73/87/201,91/201/439/444, 99, 106, 107, 107/208/320/364/428,107/247/249/252/254/364, 107/247/252, 107/247/428, 109, 159, 169, 171,172, 177, 179, 190, 190/208/247/252/428, 222, 233, 233/269,247/249/252/254/320/428, 249/252/253/254, 249/252/254/428, 251,252/253/254, 253/320, 259, 264, 289, 296, 300, 308, 308/327/439, 317,318, 320, 320/364/428, 320/428, 347, 404, 408, 417, 424, 427, 428,428/434, 431, 435, and 438, wherein the positions are numbered withreference to SEQ ID NO: 5290. In some embodiments, the polypeptidesequence of the engineered glycosyltransferase comprises at least onemutation or mutation set at one or more positions selected from 2L, 2N,7P, 25L/61S/208N/252E/253G, 25L/61S/428K, 25L/107T/208E/320I/428K,25L/208E/247L/252E/253G/364S/428K, 25L/247L/249I/252E/364S/428K, 38Q,53R/54V/55T/1535/201P, 53V/54V/308L/392H, 53V/171P/308L/392H, 54A,61S/107T/208N/252E/253G/254M/364S/428K, 61S/208W/252E/254M/428K, 64S,68K, 73S/87M/201P, 91T/201P/439D/444H, 99P, 106T, 107A,107A/208N/320I/364S/428K, 107A/247A/249I/252E/254M/364S, 107A/247A/252E,107A/247A/428K, 109, 159L, 169C, 169E, 169L, 169Q, 169V, 1715, 171T,171V, 172H, 172N, 177P, 179A, 1795, 190R, 190R/208N/247L/252E/428K,222A, 233C, 233G, 233K, 233L, 233M, 233Q, 233Q/269R, 233V,247A/249I/252E/254M/320I/428K, 249I/252E/253G/254M, 249I/252E/254M/428K,251L, 252E/253G/254M, 253G/320I, 259T, 264A, 289S, 296A, 296H, 296Q,300G, 308L, 308L/327L/439D, 317R, 318T, 320I, 320I/3645/428K, 320I/428K,347H, 347K, 347P, 347R, 404T, 408R, 417P, 424A, 424W, 427L, 427R,428E/434N, 428F, 428I, 428K, 428Q, 428R, 428S, 431E, 431R, 435C, 435K,435M, 435Q, 435R, 435T, and 438Q, wherein the positions are numberedwith reference to SEQ ID NO: 5290. In some embodiments, the polypeptidesequence of the engineered glycosyltransferase comprises at least onemutation or mutation set at one or more positions selected from E2L,E2N, T7P, I25L/T61S/K208N/S252E/S253G, I25L/T61S/V428K,I25L/L107T/K208E/V320I/V428K, I25L/K208E/K247L/S252E/S253G/G364S/V428K,I25L/K247L/Y249I/S252E/G364S/V428K, R38Q, K53R/T54V/S55T/A153S/E201P,K53V/T54V/R308L/K392H, K53V/K171P/R308L/K392H, T54A,T61S/L107T/K208N/S252E/S253G/S254M/G364S/V428K,T61S/K208W/S252E/S254M/V428K, F64S, N68K, E73S/W87M/E201P,I91T/E201P/K439D/Y444H, E99P, S106T, L107A,L107A/K208N/V320I/G364S/V428K, L107A/K247A/Y249I/S252E/S254M/G364S,L107A/K247A/S252E, L107A/K247A/V428K, R109S, N159L, G169C, G169E, G169L,G169Q, G169V, K171S, K171T, K171V, T172H, T172N, Q177P, V179A, V179S,K190R, K190R/K208N/K247L/S252E/V428K, K222A, W233C, W233G, W233K, W233L,W233M, W233Q, W233Q/Q269R, W233V, K247A/Y249I/S252E/S254M/V320I/V428K,Y249I/S252E/S253G/S254M, Y249I/S252E/S254M/V428K, A251L,S252E/S253G/S254M, S253G/V320I, E259T, F264A, K289S, R296A, R296H,R296Q, D300G, R308L, R308L/F327L/K439D, S317R, E318T, V320I,V320I/G364S/V428K, V320I/V428K, D347H, D347K, D347P, D347R, G404T,N408R, R417P, Q424A, Q424W, A427L, A427R, V428E/D434N, V428F, V428I,V428K, V428Q, V428R, V428S, D431E, D431R, V435C, V435K, V435M, V435Q,V435R, V435T, and A438Q, wherein the positions are numbered withreference to SEQ ID NO: 5290. In some embodiments, the polypeptidesequence of the engineered glycosyltransferase comprises a sequence atleast 90% identical to any of SEQ ID NOS: 5344, 5346, 5348, 5350, 5352,5354, 5356, 5358, 5360, 5362, 5364, 5366, 5368, 5370, 5372, 5374, 5376,5378, 5380, 5382, 5384, 5386, 5388, 5390, 5392, 5394, 5396, 5398, 5400,5402, 5404, 5406, 5408, 5410, 5412, 5414, 5416, 5418, 5420, 5422, 5424,5426, 5428, 5430, 5432, 5434, 5436, 5438, 5440, 5442, 5444, 5446, 5448,5450, 5452, 5454, 5456, 5458, 5460, 5462, 5464, 5466, 5468, 5470, 5472,5474, 5476, 5478, 5480, 5482, 5484, 5486, 5488, 5490, 5492, 5494, 5496,5498, 5500, 5502, 5504, 5506, 5508, 5510, 5512, 5514, 5516, 5518, 5520,5522, 5524, 5526, 5528, 5530, 5532, 5534, 5536, 5538, 5540, and 5542. Insome embodiments, the polypeptide sequence of the engineeredglycosyltransferase comprises a sequence at least 95% identical to anyof SEQ ID NOS: 5344, 5346, 5348, 5350, 5352, 5354, 5356, 5358, 5360,5362, 5364, 5366, 5368, 5370, 5372, 5374, 5376, 5378, 5380, 5382, 5384,5386, 5388, 5390, 5392, 5394, 5396, 5398, 5400, 5402, 5404, 5406, 5408,5410, 5412, 5414, 5416, 5418, 5420, 5422, 5424, 5426, 5428, 5430, 5432,5434, 5436, 5438, 5440, 5442, 5444, 5446, 5448, 5450, 5452, 5454, 5456,5458, 5460, 5462, 5464, 5466, 5468, 5470, 5472, 5474, 5476, 5478, 5480,5482, 5484, 5486, 5488, 5490, 5492, 5494, 5496, 5498, 5500, 5502, 5504,5506, 5508, 5510, 5512, 5514, 5516, 5518, 5520, 5522, 5524, 5526, 5528,5530, 5532, 5534, 5536, 5538, 5540, and 5542. In some embodiments, thepolypeptide sequence of the engineered glycosyltransferase comprises anyof SEQ ID NOS: 5344, 5346, 5348, 5350, 5352, 5354, 5356, 5358, 5360,5362, 5364, 5366, 5368, 5370, 5372, 5374, 5376, 5378, 5380, 5382, 5384,5386, 5388, 5390, 5392, 5394, 5396, 5398, 5400, 5402, 5404, 5406, 5408,5410, 5412, 5414, 5416, 5418, 5420, 5422, 5424, 5426, 5428, 5430, 5432,5434, 5436, 5438, 5440, 5442, 5444, 5446, 5448, 5450, 5452, 5454, 5456,5458, 5460, 5462, 5464, 5466, 5468, 5470, 5472, 5474, 5476, 5478, 5480,5482, 5484, 5486, 5488, 5490, 5492, 5494, 5496, 5498, 5500, 5502, 5504,5506, 5508, 5510, 5512, 5514, 5516, 5518, 5520, 5522, 5524, 5526, 5528,5530, 5532, 5534, 5536, 5538, 5540, and 5542.

The present invention also provides engineered glycosyltransferases,wherein the polypeptide sequences of the engineered glycosyltransferasescomprise at least one mutation or mutation set at one or more positionsselected from 2, 49, 73, 87, 87/88, 87/88/94/159/177/208, 87/88/159/198,87/88/177/198/249, 87/88/208, 87/159/177, 87/159/320, 87/177/249,87/198, 87/198/199, 87/198/208/320, 87/208, 87/208/320, 87/320, 88,88/177, 88/177/208, 88/177/320, 88/198, 88/199, 88/199/208, 88/208,88/208/249/320, 88/320, 113, 134, 135, 158, 159/177/198, 159/177/208,159/198/208/320, 171, 173, 177/198, 177/208, 195, 214, 222, 253, 256,257, 268, 272, 289, 300, 302, 330, 348, 374, 392, 399, 408, 411, 412,437, 439, 445, and 453, wherein the positions are numbered withreference to SEQ ID NO: 5372. In some embodiments, the polypeptidesequence of the engineered glycosyltransferase comprises at least onemutation or mutation set at one or more positions selected from 2G, 2H,2P, 49A, 73P, 73T, 87M, 87M/88R, 87M/88R/94K/159Q/177P/208E,87M/88R/159Q/198E, 87M/88R/177P/198E/249I, 87M/88R/208E, 87M/159Q/177P,87M/159Q/320I, 87M/177P/249I, 87M/198E, 87M/198E/199K,87M/198E/208E/320I, 87M/208E, 87M/208E/320I, 87M/320I, 88R, 88R/177P,88R/177P/208E, 88R/177P/320I, 88R/198I, 88R/199K, 88R/199K/208E,88R/208E, 88R/208E/249I/320I, 88R/320I, 113S, 134G, 134S, 135A, 158D,158E, 159Q/177P/198E, 159Q/177P/208E, 159Q/198I/208E/320I, 171T, 173N,173P, 177P/198E, 177P/208E, 195H, 214L, 222R, 253G, 256P, 257A, 257Q,268A, 268G, 268H, 272K, 289T, 300G, 302R, 330D, 348C, 374K, 374R, 392D,399D, 408D, 411Q, 412H, 437I, 437T, 437V, 439A, 445T, and 453R, whereinthe positions are numbered with reference to SEQ ID NO: 5372. In someembodiments, the polypeptide sequence of the engineeredglycosyltransferase comprises at least one mutation or mutation set atone or more positions selected from E2G, E2H, E2P, F49A, E73P, E73T,W87M, W87M/I88R, W87M/I88R/E94K/N159Q/Q177P/K208E,W87M/I88R/N159Q/D198E, W87M/I88R/Q177P/D198E/Y249I, W87M/I88R/K208E,W87M/N159Q/Q177P, W87M/N159Q/V320I, W87M/Q177P/Y249I, W87M/D198E,W87M/D198E/A199K, W87M/D198E/K208E/V320I, W87M/K208E, W87M/K208E/V320I,W87M/V320I, I88R, I88R/Q177P, I88R/Q177P/K208E, I88R/Q177P/V320I,I88R/D198I, I88R/A199K, I88R/A199K/K208E, I88R/K208E,I88R/K208E/Y249I/V320I, I88R/V320I, E113S, D134G, D134S, S135A, P158D,P158E, N159Q/Q177P/D198E, N159Q/Q177P/K208E, N159Q/D198I/K208E/V320I,K171T, R173N, R173P, Q177P/D198E, Q177P/K208E, N195H, S214L, K222R,S253G, L256P, L257A, L257Q, D268A, D268G, D268H, P272K, K289T, D300G,K302R, E330D, A348C, S374K, S374R, K392D, N399D, N408D, R411Q, R412H,L437I, L437T, L437V, K439A, E445T, and Y453R, wherein the positions arenumbered with reference to SEQ ID NO: 5372. In some embodiments, thepolypeptide sequence of the engineered glycosyltransferase comprises asequence at least 90% identical to any of SEQ ID NOS: 5544, 5546, 5548,5550, 5552, 5554, 5556, 5558, 5560, 5562, 5564, 5566, 5568, 5570, 5572,5574, 5576, 5578, 5580, 5582, 5584, 5586, 5588, 5590, 5592, 5594, 5596,5598, 5600, 5602, 5604, 5606, 5608, 5610, 5612, 5614, 5616, 5618, 5620,5622, 5624, 5626, 5628, 5630, 5632, 5634, 5636, 5638, 5640, 5642, 5644,5646, 5648, 5650, 5652, 5654, 5656, 5658, 5660, 5662, 5664, 5666, 5668,5670, 5672, 5674, 5676, 5678, 5680, 5682, 5684, 5686, 5688, and 5690. Insome embodiments, the polypeptide sequence of the engineeredglycosyltransferase comprises a sequence at least 95% identical to anyof SEQ ID NOS: 5544, 5546, 5548, 5550, 5552, 5554, 5556, 5558, 5560,5562, 5564, 5566, 5568, 5570, 5572, 5574, 5576, 5578, 5580, 5582, 5584,5586, 5588, 5590, 5592, 5594, 5596, 5598, 5600, 5602, 5604, 5606, 5608,5610, 5612, 5614, 5616, 5618, 5620, 5622, 5624, 5626, 5628, 5630, 5632,5634, 5636, 5638, 5640, 5642, 5644, 5646, 5648, 5650, 5652, 5654, 5656,5658, 5660, 5662, 5664, 5666, 5668, 5670, 5672, 5674, 5676, 5678, 5680,5682, 5684, 5686, 5688, and 5690. In some embodiments, the polypeptidesequence of the engineered glycosyltransferase comprises any of SEQ IDNOS: 5544, 5546, 5548, 5550, 5552, 5554, 5556, 5558, 5560, 5562, 5564,5566, 5568, 5570, 5572, 5574, 5576, 5578, 5580, 5582, 5584, 5586, 5588,5590, 5592, 5594, 5596, 5598, 5600, 5602, 5604, 5606, 5608, 5610, 5612,5614, 5616, 5618, 5620, 5622, 5624, 5626, 5628, 5630, 5632, 5634, 5636,5638, 5640, 5642, 5644, 5646, 5648, 5650, 5652, 5654, 5656, 5658, 5660,5662, 5664, 5666, 5668, 5670, 5672, 5674, 5676, 5678, 5680, 5682, 5684,5686, 5688, and 5690.

The present invention also provides engineered glycosyltransferases,wherein the polypeptide sequences of the engineered glycosyltransferasescomprise at least one mutation or mutation set at one or more positionsselected from 2/7/107/233/252/253/320, 2/7/107/233/252/253/320/408/428,2/7/233/252/253/320/427/428, 2/233/252/317/320,7/107/190/233/252/253/317/408/427, 7/107/233/252/253,7/107/233/252/317/320, 7/107/233/252/317/408, 7/190/233/252/253/320/427,7/190/233/252/317/320/427/428, 7/190/233/252/408/427,7/233/252/253/317/408/427, 7/233/252/253/408, 7/233/252/317/320/427,7/233/252/317/428, 64/169/201/347/392, 64/172/264/268/347/392/417,91/94/171/172/201/264/347, 91/201/264/347/392, 94/201/264/347/435,107/190/233/252, 107/190/233/252/317/320, 107/233/252/253, 107/252/317,169/171/172/264/392/435, 169/171/201/264/392/435, 169/172/201/264/347,169/172/201/264/347/392/435, 169/172/201/347/392, 169/172/201/417/435,171/172/201/264/392/417/435, 171/201/392/417, 190/233/252,190/233/252/253/317/320/408/428, 190/233/252/317/408,190/233/252/320/408, 201/264/347/392/417/435, 201/264/347/392/435,201/264/347/417/435, 233/252/253/317/320/427/428, 233/252/253/408/427,233/252/317/408, 233/252/317/427, 233/252/320, 233/252/320/408/428,348/374/435, and 374, wherein the positions are numbered with referenceto SEQ ID NO: 5562. In some embodiments, the polypeptide sequence of theengineered glycosyltransferase comprises at least one mutation ormutation set at one or more positions selected from2L/7P/233K/252E/253G/320I/427R/428R, 2N/7P/107A/233K/252E/253G/320I,2N/7P/107A/233K/252E/253G/320I/408R/428R, 2N/233K/252E/317R/320I,7P/107A/190R/233K/252E/253G/317R/408R/427R, 7P/107A/233K/252E/253G,7P/107A/233K/252E/317R/320I, 7P/107A/233K/252E/317R/408R,7P/190R/233K/252E/253G/320I/427L, 7P/190R/233K/252E/317R/320I/427R/428R,7P/190R/233K/252E/408R/427R, 7P/233K/252E/253G/317R/408R/427L,7P/233K/252E/253G/408R, 7P/233K/252E/317R/320I/427L,7P/233K/252E/317R/428R, 64S/169E/201P/347P/392H,64S/172H/264A/268V/347P/392H/417P, 91T/94E/171T/172H/201P/264A/347G,91T/201P/264A/347G/392H, 94E/201P/264A/347G/435Q, 107A/190R/233K/252E,107A/190R/233K/252E/317R/320I, 107A/233K/252E/253G, 107A/252E/317R,169E/171T/172H/264A/392H/435Q, 169E/171T/201P/264A/392H/435Q,169E/172H/201P/264A/347G, 169E/172H/201P/264A/347K/392H/435R,169E/172H/201P/347G/392H, 169E/172H/201P/417P/435R,171T/172H/201P/264A/392H/417P/435R, 171T/201P/392H/417P, 190R/233K/252E,190R/233K/252E/253G/317R/320I/408R/428R, 190R/233K/252E/317R/408R,190R/233K/252E/320I/408R, 201P/264A/347K/392H/417P/435R,201P/264A/347K/417P/435R, 201P/264A/347P/392H/435Q,233K/252E/253G/317R/320I/427R/428R, 233K/252E/253G/408R/427L,233K/252E/317R/408R, 233K/252E/317R/427R, 233K/252E/320I,233K/252E/320I/408R/428R, 348S/374R/435R, and 374R, wherein thepositions are numbered with reference to SEQ ID NO: 5562. In someembodiments, the polypeptide sequence of the engineeredglycosyltransferase comprises at least one mutation or mutation set atone or more positions selected fromE2L/T7P/W233K/S252E/S253G/V320I/A427R/K428R,E2N/T7P/L107A/W233K/S252E/S253G/V320I,E2N/T7P/L107A/W233K/S252E/S253G/V320I/N408R/K428R,E2N/W233K/S252E/S317R/V320I,T7P/L107A/K190R/W233K/S252E/S253G/S317R/N408R/A427R,T7P/L107A/W233K/S252E/S253G, T7P/L107A/W233K/S252E/S317R/V320I,T7P/L107A/W233K/S252E/S317R/N408R,T7P/K190R/W233K/S252E/S253G/V320I/A427L,T7P/K190R/W233K/S252E/S317R/V320I/A427R/K428R,T7P/K190R/W233K/S252E/N408R/A427R,T7P/W233K/S252E/S253G/S317R/N408R/A427L, T7P/W233K/S252E/S253G/N408R,T7P/W233K/S252E/S317R/V320I/A427L, T7P/W233K/S252E/S317R/K428R,F64S/G169E/E201P/D347P/K392H, F64S/T172H/F264A/D268V/D347P/K392H/R417P,I91T/K94E/K171T/T172H/E201P/F264A/D347G, I91T/E201P/F264A/D347G/K392H,K94E/E201P/F264A/D347G/V435Q, L107A/K190R/W233K/S252E,L107A/K190R/W233K/S252E/S317R/V320I, L107A/W233K/S252E/S253G,L107A/S252E/S317R, G169E/K171T/T172H/F264A/K392H/V435Q,G169E/K171T/E201P/F264A/K392H/V435Q, G169E/T172H/E201P/F264A/D347G,G169E/T172H/E201P/F264A/D347K/K392H/V435R,G169E/T172H/E201P/D347G/K392H, G169E/T172H/E201P/R417P/V435R,K171T/T172H/E201P/F264A/K392H/R417P/V435R, K171T/E201P/K392H/R417P,K190R/W233K/S252E, K190R/W233K/S252E/S253G/S317R/V320I/N408R/K428R,K190R/W233K/S252E/S317R/N408R, K190R/W233K/S252E/V320I/N408R,E201P/F264A/D347K/K392H/R417P/V435R, E201P/F264A/D347K/R417P/V435R,E201P/F264A/D347P/K392H/V435Q,W233K/S252E/S253G/S317R/V320I/A427R/K428R,W233K/S252E/S253G/N408R/A427L, W233K/S252E/S317R/N408R,W233K/S252E/S317R/A427R, W233K/S252E/V320I,W233K/S252E/V320I/N408R/K428R, A348S/S374R/V435R, and S374R, wherein thepositions are numbered with reference to SEQ ID NO: 5562. In someembodiments, the polypeptide sequence of the engineeredglycosyltransferase comprises at least one mutation or mutation set atone or more positions selected from 14, 55, 56, 255, 282, 308, 336, 342,364, 391, 407, and 422, wherein the positions are numbered withreference to SEQ ID NO: 5562. In some embodiments, the polypeptidesequence of the engineered glycosyltransferase comprises at least onemutation or mutation set at one or more positions selected from 14I,55V, 56A, 255L, 2825, 308L, 308Q, 336Q, 342W, 364A, 3645, 391C, 407C,407V, and 422Q, wherein the positions are numbered with reference to SEQID NO: 5562. In some embodiments, the polypeptide sequence of theengineered glycosyltransferase comprises at least one mutation ormutation set at one or more positions selected from V14I, S55V, L56A,S255L, T282S, R308L, R308Q, K336Q, E342W, G364A, G364S, L391C, E407C,E407V, and M422Q, wherein the positions are numbered with reference toSEQ ID NO: 5562. In some embodiments, the polypeptide sequence of theengineered glycosyltransferase comprises a sequence at least 90%identical to any of SEQ ID NOS: 5692, 5694, 5696, 5698, 5700, 5702,5704, 5706, 5708, 5710, 5712, 5714, 5716, 5718, 5720, 5722, 5724, 5726,5728, 5730, 5732, 5734, 5736, 5738, 5740, 5742, 5744, 5746, 5748, 5750,5752, 5754, 5756, 5758, 5760, 5762, 5764, 5766, 5768, 5770, 5772, 5774,5776, 5778, 5780, 5782, 5784, 5786, 5788, 5790, 5792, 5794, 5796, 5798,5800, 5802, 5804, 5806, 5808, 5810, 5812, and 5814. In some embodiments,the polypeptide sequence of the engineered glycosyltransferase comprisesa sequence at least 95% identical to any of SEQ ID NOS: 5692, 5694,5696, 5698, 5700, 5702, 5704, 5706, 5708, 5710, 5712, 5714, 5716, 5718,5720, 5722, 5724, 5726, 5728, 5730, 5732, 5734, 5736, 5738, 5740, 5742,5744, 5746, 5748, 5750, 5752, 5754, 5756, 5758, 5760, 5762, 5764, 5766,5768, 5770, 5772, 5774, 5776, 5778, 5780, 5782, 5784, 5786, 5788, 5790,5792, 5794, 5796, 5798, 5800, 5802, 5804, 5806, 5808, 5810, 5812, and5814. In some embodiments, the polypeptide sequence of the engineeredglycosyltransferase comprises any of SEQ ID NOS: 5692, 5694, 5696, 5698,5700, 5702, 5704, 5706, 5708, 5710, 5712, 5714, 5716, 5718, 5720, 5722,5724, 5726, 5728, 5730, 5732, 5734, 5736, 5738, 5740, 5742, 5744, 5746,5748, 5750, 5752, 5754, 5756, 5758, 5760, 5762, 5764, 5766, 5768, 5770,5772, 5774, 5776, 5778, 5780, 5782, 5784, 5786, 5788, 5790, 5792, 5794,5796, 5798, 5800, 5802, 5804, 5806, 5808, 5810, 5812, and 5814.

The present invention also provides engineered glycosyltransferases,wherein the polypeptide sequences of the engineered glycosyltransferasescomprise at least one mutation or mutation set at one or more positionsselected from 2/4/113/158/163/302/364/399/449, 2/4/113/158/163/330/449,2/4/113/163/449, 2/4/158/163/364/449, 2/4/158/222/257/302/330,2/4/158/330, 2/4/163/222/302/330/364/449, 2/4/163/257,2/4/163/257/330/399/449, 2/4/163/330/449, 2/4/163/364, 2/4/163/364/453,2/4/302, 2/113, 2/113/163, 2/113/163/449, 2/113/330/399, 2/113/449,2/158/163, 2/158/163/364/399, 2/163/364/453, 2/364/449,4/113/158/163/330/364/399, 4/113/158/302/330/364, 4/113/163/364/399,4/158/163/364/399, 4/364/449, 87, 87/95/198, 87/198, 87/266, 87/322,107/134/135/195/412/417, 107/195/268/322/439, 107/195/272,107/195/417/439, 107/374/417/439, 113/158/163/364/399, 134,134/135/195/268/317, 158/163/257/330/364/449, 158/163/302/330/364/399,158/163/364, 163/257/302/364, 163/302/330/364/449, 163/364, 173,173/190/233/252/427/437, 173/190/252/257/347/427/437,173/190/257/374/437, 173/190/257/427/437, 173/233/252/257/427,173/233/437, 173/252, 173/252/268/437, 173/252/347/411, 173/257/374/437,173/374/437, 173/427/437, 190/233/252/257/347/411/437, 190/252,190/252/257/285/427, 190/252/257/411/437, 190/252/257/427,190/374/427/437, 195/272/320/439, 195/317/320, 198, 198/244, 198/292,233/252, 233/252/257, 233/252/257/347, 233/252/257/347/411/437,233/252/257/427/437, 233/252/285, 233/252/285/437, 233/252/411/437,233/252/437, 233/374, 233/374/437, 252/257, 252/257/347/437,257/347/411/437, 268/417, 285/347/437, 302/364, 330/364, 347/411/437,347/427/437, 364/399, 374, 374/411/437, 408/417, 411/437, and 437,wherein the positions are numbered with reference to SEQ ID NO:5708. Insome embodiments, the polypeptide sequence of the engineeredglycosyltransferase comprises at least one mutation or mutation set atone or more positions selected from 2P/4T/113S/158D/163A/330D/449R,2P/4T/113S/158D/163R/302R/364S/399D/449R, 2P/4T/113S/163R/449R,2P/4T/158D/163A/364S/449R, 2P/4T/158D/222R/257Q/302R/330D,2P/4T/158D/330D, 2P/4T/163A/257Q, 2P/4T/163A/330D/449R, 2P/4T/163A/364S,2P/4T/163A/364S/453R, 2P/4T/163R/222R/302R/330D/364S/449R,2P/4T/163R/257Q/330D/399D/449R, 2P/4T/302R, 2P/113S, 2P/113S/163A,2P/113S/163R/449R, 2P/113S/330D/399D, 2P/113S/449R,2P/158D/163A/364S/399D, 2P/158D/163R, 2P/163R/364S/453R, 2P/364S/449R,4T/113S/158D/163A/330D/364S/399D, 4T/113S/158D/302R/330D/364S,4T/113S/163A/364S/399D, 4T/158D/163A/364S/399D, 4T/364S/449R, 87W,87W/95L/198E, 87W/198E, 87W/198I, 87W/266L, 87W/322S,107A/134S/135A/195H/412H/417P, 107A/195H/268A/322L/439P, 107A/195H/272K,107A/195H/417P/439P, 107A/374T/417P/439P, 113S/158D/163A/364S/399D,113S/158D/163R/364S/399D, 1345, 134S/135A/195H/268A/317R,158D/163A/257Q/330D/364S/449R, 158D/163A/364S,158D/163R/302R/330D/364S/399D, 163A/257Q/302R/364S, 163A/364S,163R/302R/330D/364S/449R, 173N, 173N/190R/233K/252E/427R/437V,173N/190R/252E/257A/347G/427R/437I, 173N/190R/257A/374K/437V,173N/190R/257A/427R/437I, 173N/233K/252E/257A/427R, 173N/233K/437I,173N/252E, 173N/252E/268H/437V, 173N/252E/347G/411Q,173N/257A/374K/437V, 173N/374K/437I, 173N/427R/437I,190R/233K/252E/257A/347G/411Q/437I, 190R/252E, 190R/252E/257A/285Q/427R,190R/252E/257A/411Q/437I, 190R/252E/257A/427R, 190R/374K/427R/437V,195H/272K/320I/439P, 195H/317R/320I, 198E, 198E/292P, 198I, 198I/244L,233K/252E, 233K/252E/257A, 233K/252E/257A/347G,233K/252E/257A/347G/411Q/437I, 233K/252E/257A/427R/437V, 233K/252E/285Q,233K/252E/285Q/437V, 233K/252E/411Q/437V, 233K/252E/437V, 233K/374K,233K/374K/437I, 252E/257A, 252E/257A/347G/437V, 257A/347G/411Q/437V,268A/417P, 285Q/347G/437V, 302R/364S, 330D/364S, 347G/411Q/437I,347G/427R/437V, 364S/399D, 374K, 374K/411Q/437I, 374K/411Q/437V,408D/417P, 411Q/437V, and 437, wherein the positions are numbered withreference to SEQ ID NO:5708. In some embodiments, the polypeptidesequence of the engineered glycosyltransferase comprises at least onemutation or mutation set at one or more positions selected fromE2P/K4T/E113S/P158D/L163A/E330D/S449R,E2P/K4T/E113S/P158D/L163R/K302R/G364S/N399D/S449R,E2P/K4T/E113S/L163R/S449R, E2P/K4T/P158D/L163A/G364S/S449R,E2P/K4T/P158D/K222R/L257Q/K302R/E330D, E2P/K4T/P158D/E330D,E2P/K4T/L163A/L257Q, E2P/K4T/L163A/E330D/S449R, E2P/K4T/L163A/G364S,E2P/K4T/L163A/G364S/Y453R, E2P/K4T/L163R/K222R/K302R/E330D/G364S/S449R,E2P/K4T/L163R/L257Q/E330D/N399D/S449R, E2P/K4T/K302R, E2P/E113S,E2P/E113S/L163A, E2P/E113S/L163R/S449R, E2P/E113S/E330D/N399D,E2P/E113S/S449R, E2P/P158D/L163A/G364S/N399D, E2P/P158D/L163R,E2P/L163R/G364S/Y453R, E2P/G364S/S449R,K4T/E113S/P158D/L163A/E330D/G364S/N399D,K4T/E113S/P158D/K302R/E330D/G364S, K4T/E113S/L163A/G364S/N399D,K4T/P158D/L163A/G364S/N399D, K4T/G364S/S449R, M87W, M87W/H95L/D198E,M87W/D198E, M87W/D198I, M87W/W266L, M87W/P322S,L107A/D134S/S135A/N195H/R412H/R417P, L107A/N195H/D268A/P322L/K439P,L107A/N195H/P272K, L107A/N195H/R417P/K439P, L107A/S374T/R417P/K439P,E113S/P158D/L163A/G364S/N399D, E113S/P158D/L163R/G364S/N399D, D134S,D134S/S135A/N195H/D268A/S317R, P158D/L163A/L257Q/E330D/G364S/S449R,P158D/L163A/G364S, P158D/L163R/K302R/E330D/G364S/N399D,L163A/L257Q/K302R/G364S, L163A/G364S, L163R/K302R/E330D/G364S/S449R,R173N, R173N/K190R/W233K/S252E/A427R/L437V,R173N/K190R/S252E/L257A/K347G/A427R/L437I,R173N/K190R/L257A/S374K/L437V, R173N/K190R/L257A/A427R/L437I,R173N/W233K/S252E/L257A/A427R, R173N/W233K/L437I, R173N/S252E,R173N/S252E/D268H/L437V, R173N/S252E/K347G/R411Q,R173N/L257A/S374K/L437V, R173N/S374K/L437I, R173N/A427R/L437I,K190R/W233K/S252E/L257A/K347G/R411Q/L437I, K190R/S252E,K190R/S252E/L257A/E285Q/A427R, K190R/S252E/L257A/R411Q/L437I,K190R/S252E/L257A/A427R, K190R/S374K/A427R/L437V,N195H/P272K/V320I/K439P, N195H/S317R/V320I, D198E, D198E/L292P, D198I,D198I/P244L, W233K/S252E, W233K/S252E/L257A, W233K/S252E/L257A/K347G,W233K/S252E/L257A/K347G/R411Q/L437I, W233K/S252E/L257A/A427R/L437V,W233K/S252E/E285Q, W233K/S252E/E285Q/L437V, W233K/S252E/R411Q/L437V,W233K/S252E/L437V, W233K/S374K, W233K/S374K/L437I, S252E/L257A,S252E/L257A/K347G/L437V, L257A/K347G/R411Q/L437V, D268A/R417P,E285Q/K347G/L437V, K302R/G364S, E330D/G364S, K347G/R411Q/L437I,K347G/A427R/L437V, G364S/N399D, S374K, S374K/R411Q/L437I,S374K/R411Q/L437V, N408D/R417P, R411Q/L437V, and L437I, wherein thepositions are numbered with reference to SEQ ID NO:5708. In someembodiments, the polypeptide sequence of the engineeredglycosyltransferase comprises a sequence at least 90% identical to anyof SEQ ID NOS: 5816, 5818, 5820, 5822, 5824, 5826, 5828, 5830, 5832,5834, 5836, 5838, 5840, 5842, 5844, 5846, 5848, 5850, 5852, 5854, 5856,5858, 5860, 5862, 5864, 5866, 5868, 5870, 5872, 5874, 5876, 5878, 5880,5882, 5884, 5886, 5888, 5890, 5892, 5894, 5896, 5898, 5900, 5902, 5904,5906, 5908, 5910, 5912, 5914, 5916, 5918, 5920, 5922, 5924, 5926, 5928,5930, 5932, 5934, 5936, 5938, 5940, 5942, 5944, 5946, 5948, 5950, 5952,5954, 5956, 5958, 5960, 5962, 5964, 5966, 5968, 5970, 5972, 5974, 5976,5978, 5980, 5982, 5984, 5986, 5988, 5990, 5992, 5994, 5996, 5998, 6000,6002, 6004, 6006, 6008, 6010, 6012, and 6014. In some embodiments, thepolypeptide sequence of the engineered glycosyltransferase comprises asequence at least 95% identical to any of SEQ ID NOS: 5816, 5818, 5820,5822, 5824, 5826, 5828, 5830, 5832, 5834, 5836, 5838, 5840, 5842, 5844,5846, 5848, 5850, 5852, 5854, 5856, 5858, 5860, 5862, 5864, 5866, 5868,5870, 5872, 5874, 5876, 5878, 5880, 5882, 5884, 5886, 5888, 5890, 5892,5894, 5896, 5898, 5900, 5902, 5904, 5906, 5908, 5910, 5912, 5914, 5916,5918, 5920, 5922, 5924, 5926, 5928, 5930, 5932, 5934, 5936, 5938, 5940,5942, 5944, 5946, 5948, 5950, 5952, 5954, 5956, 5958, 5960, 5962, 5964,5966, 5968, 5970, 5972, 5974, 5976, 5978, 5980, 5982, 5984, 5986, 5988,5990, 5992, 5994, 5996, 5998, 6000, 6002, 6004, 6006, 6008, 6010, 6012,and 6014. In some embodiments, the polypeptide sequence of theengineered glycosyltransferase comprises any of SEQ ID NOS: 5816, 5818,5820, 5822, 5824, 5826, 5828, 5830, 5832, 5834, 5836, 5838, 5840, 5842,5844, 5846, 5848, 5850, 5852, 5854, 5856, 5858, 5860, 5862, 5864, 5866,5868, 5870, 5872, 5874, 5876, 5878, 5880, 5882, 5884, 5886, 5888, 5890,5892, 5894, 5896, 5898, 5900, 5902, 5904, 5906, 5908, 5910, 5912, 5914,5916, 5918, 5920, 5922, 5924, 5926, 5928, 5930, 5932, 5934, 5936, 5938,5940, 5942, 5944, 5946, 5948, 5950, 5952, 5954, 5956, 5958, 5960, 5962,5964, 5966, 5968, 5970, 5972, 5974, 5976, 5978, 5980, 5982, 5984, 5986,5988, 5990, 5992, 5994, 5996, 5998, 6000, 6002, 6004, 6006, 6008, 6010,6012, and 6014.

The present invention also provides engineered glycosyltransferases,wherein the polypeptide sequences of the engineered glycosyltransferasescomprise at least one mutation or mutation set at one or more positionsselected from 2/4/14/158/282/364/449, 2/4/14/158/282/407,2/4/14/163/198, 2/4/14/163/282/399, 2/4/14/198/308/407,2/4/14/282/308/364, 2/4/14/282/308/407, 2/4/14/282/364,2/4/158/163/364/399/407, 2/4/158/198/282/364/407,2/4/163/198/282/308/342, 2/4/163/282/342/364, 2/4/163/364/399/449,2/4/198/282/449, 2/4/198/364/391/449, 2/4/282/342/364/407,2/14/158/198/407/449, 2/14/163/308/364, 2/14/163/364/407, 2/14/282,2/14/282/308, 2/14/282/308/364, 2/14/282/399, 2/14/308, 2/14/308/364,2/158/163/407/449, 2/163/282, 2/163/282/308/364, 2/198/282/308/342,2/198/282/399, 2/282, 2/282/308/342/407, 2/282/308/391/407, 2/282/399,2/282/399/407, 2/308, 2/308/364/399, 4/14/158/163/198/282/407,4/14/158/282/364/391/407, 4/14/163/282, 4/14/163/282/308/342/407/449,4/14/198/308/364, 4/14/282, 4/14/282/308/364/407/449,4/14/282/342/399/407, 4/14/364/391, 4/158/282/364/399,4/163/282/308/407, 4/198/399/407, 4/282/342/364/407, 4/282/364/407,4/364, 12, 14/158/163/198/364, 14/158/163/282/364,14/158/198/282/342/364/449, 14/158/364, 14/163/198/282/342/364/449,14/163/282/308, 14/282, 14/282/308, 14/282/342/364/391, 14/282/364,14/282/364/391/407/449, 14/282/399/407/449, 14/407, 37, 44, 70, 71/331,74, 75, 90, 106, 108, 112, 114, 115, 131, 138, 139, 156, 158/282/407,158/282/407/449, 158/364/399/407/449, 158/364/449, 162, 163/198/282/342,163/282/308/364/399, 163/282/399/407, 163/282/407/449, 174,198/282/308/342, 198/282/407, 198/308, 247, 248, 254, 258,282/308/342/364/449, 282/308/449, 365, 389, 401/402, 416, 427, 429, 432,433, and 456, wherein the positions are numbered with reference to SEQID NO: 5976. In some embodiments, the polypeptide sequence of theengineered glycosyltransferase comprises at least one mutation ormutation set at one or more positions selected from2P/4T/14I/158D/282S/364A/449R, 2P/4T/14I/158D/282S/407V,2P/4T/14I/163A/198E, 2P/4T/14I/163A/282S/399D, 2P/4T/14I/198E/308L/407V,2P/4T/14I/282S/308L/364A, 2P/4T/14I/282S/308L/407V, 2P/4T/14I/282S/364A,2P/4T/158D/163A/364A/399D/407V, 2P/4T/158D/198E/282S/364A/407V,2P/4T/163A/198E/282S/308L/342W, 2P/4T/163A/282S/342W/364A,2P/4T/163A/364A/399D/449R, 2P/4T/198E/282S/449R,2P/4T/198E/364A/391C/449R, 2P/4T/282S/342W/364A/407V,2P/14I/158D/198E/407V/449R, 2P/14I/163A/308L/364A,2P/14I/163A/364A/407V, 2P/14I/2825, 2P/14I/282S/308L,2P/14I/282S/308L/364A, 2P/14I/282S/399D, 2P/14I/308L, 2P/14I/308L/364A,2P/158D/163A/407V/449R, 2P/163A/282S, 2P/163A/282S/308L/364A,2P/198E/282S/308L/342W, 2P/198E/282S/399D, 2P/282S,2P/282S/308L/342W/407V, 2P/282S/308L/391C/407V, 2P/282S/399D,2P/282S/399D/407V, 2P/308L, 2P/308L/364A/399D,4T/14I/158D/163A/198E/282S/407V, 4T/14I/158D/282S/364A/391C/407V,4T/14I/163A/282S, 4T/141/163A/282S/308L/342W/407V/449R,4T/141/198E/308L/364A, 4T/141/282S, 4T/14I/282S/308L/364A/407V/449R,4T/14I/282S/342W/399D/407V, 4T/14I/364A/391C, 4T/158D/282S/364A/399D,4T/163A/282S/308L/407V, 4T/198E/399D/407V, 4T/282S/342W/364A/407V,4T/282S/364A/407V, 4T/364A, 12S, 14I/158D/163A/198E/364A,14I/158D/163A/282S/364A, 14I/158D/198E/282S/342W/364A/449R,14I/158D/364A, 141/163A/198E/282S/342W/364A/449R, 14I/163A/282S/308L,14I/282S, 14I/282S/308L, 14I/282S/342W/364A/391C, 14I/282S/364A,14I/282S/364A/391C/407V/449R, 14I/282S/399D/407V/449R, 14I/407V, 37R,44V, 70K, 71L/331K, 74H, 75G, 75M, 90Q, 905, 90T, 106Y, 108H, 112N,114P, 115R, 131V, 138V, 139A, 139R, 139, 156C, 158D/282S/407V,158D/282S/407V/449R, 158D/364A/399D/407V/449R, 158D/364A/449R, 162A,163A/198E/282S/342W, 163A/282S/308L/364A/399D, 163A/282S/399D/407V,163A/282S/407V/449R, 174P, 198E/282S/308L/342W, 198E/282S/407V,198E/308L, 247C, 247L, 248C, 248L, 248W, 254Q, 258N,282S/308L/342W/364A/449R, 282S/308L/449R, 365I, 389A, 389E, 389S,401F/402L, 416L, 4165, 427K, 427R, 429W, 432L, 433L, and 456R, whereinthe positions are numbered with reference to SEQ ID NO: 5976. In someembodiments, the polypeptide sequence of the engineeredglycosyltransferase comprises at least one mutation or mutation set atone or more positions selected fromE2P/K4T/V14I/P158D/T282S/G364A/S449R, E2P/K4T/V14I/P158D/T282S/E407V,E2P/K4T/V14I/L163A/D198E, E2P/K4T/V14I/L163A/T282S/N399D,E2P/K4T/V14I/D198E/R308L/E407V, E2P/K4T/V14I/T282S/R308L/G364A,E2P/K4T/V14I/T282S/R308L/E407V, E2P/K4T/V14I/T282S/G364A,E2P/K4T/P158D/L163A/G364A/N399D/E407V,E2P/K4T/P158D/D198E/T282S/G364A/E407V,E2P/K4T/L163A/D198E/T282S/R308L/E342W, E2P/K4T/L163A/T282S/E342W/G364A,E2P/K4T/L163A/G364A/N399D/S449R, E2P/K4T/D198E/T282S/S449R,E2P/K4T/D198E/G364A/L391C/S449R, E2P/K4T/T282S/E342W/G364A/E407V,E2P/V14I/P158D/D198E/E407V/S449R, E2P/V14I/L163A/R308L/G364A,E2P/V14I/L163A/G364A/E407V, E2P/V14I/T282S, E2P/V14I/T282S/R308L,E2P/V14I/T282S/R308L/G364A, E2P/V14I/T282S/N399D, E2P/V14I/R308L,E2P/V14I/R308L/G364A, E2P/P158D/L163A/E407V/S449R, E2P/L163A/T282S,E2P/L163A/T282S/R308L/G364A, E2P/D198E/T282S/R308L/E342W,E2P/D198E/T282S/N399D, E2P/T282S, E2P/T282S/R308L/E342W/E407V,E2P/T282S/R308L/L391C/E407V, E2P/T282S/N399D, E2P/T282S/N399D/E407V,E2P/R308L, E2P/R308L/G364A/N399D,K4T/V14I/P158D/L163A/D198E/T282S/E407V,K4T/V14I/P158D/T282S/G364A/L391C/E407V, K4T/V14I/L163A/T282S,K4T/V14I/L163A/T282S/R308L/E342W/E407V/S449R,K4T/V14I/D198E/R308L/G364A, K4T/V14I/T282S,K4T/V14I/T282S/R308L/G364A/E407V/S449R,K4T/V14I/T282S/E342W/N399D/E407V, K4T/V14I/G364A/L391C,K4T/P158D/T282S/G364A/N399D, K4T/L163A/T282S/R308L/E407V,K4T/D198E/N399D/E407V, K4T/T282S/E342W/G364A/E407V,K4T/T282S/G364A/E407V, K4T/G364A, R12S, V14I/P158D/L163A/D198E/G364A,V14I/P158D/L163A/T282S/G364A, V14I/P158D/D198E/T282S/E342W/G364A/S449R,V14I/P158D/G364A, V14I/L163A/D198E/T282S/E342W/G364A/S449R,V14I/L163A/T282S/R308L, V14I/T282S, V14I/T282S/R308L,V14I/T282S/E342W/G364A/L391C, V14I/T282S/G364A,V14I/T282S/G364A/L391C/E407V/S449R, V14I/T282S/N399D/E407V/S449R,V14I/E407V, S37R, I44V, P70K, Q71L/R331K, W74H, L75G, L75M, P90Q, P90S,P90T, S106Y, M108H, P112N, D114P, E115R, P131V, L138V, P139A, P139R,P139S, S156C, P158D/T282S/E407V, P158D/T282S/E407V/S449R,P158D/G364A/N399D/E407V/S449R, P158D/G364A/S449R, E162A,L163A/D198E/T282S/E342W, L163A/T282S/R308L/G364A/N399D,L163A/T282S/N399D/E407V, L163A/T282S/E407V/S449R, L174P,D198E/T282S/R308L/E342W, D198E/T282S/E407V, D198E/R308L, K247C, K247L,H248C, H248L, H248W, S254Q, D258N, T282S/R308L/E342W/G364A/S449R,T282S/R308L/S449R, V365I, D389A, D389E, D389S, L401F/E402L, D416L,D416S, A427K, A427R, L429W, K432L, A433L, and S456R, wherein thepositions are numbered with reference to SEQ ID NO: 5976. In someembodiments, the polypeptide sequence of the engineeredglycosyltransferase comprises a sequence at least 95% identical to anyof SEQ ID NOS: 6016, 6018, 6020, 6022, 6024, 6026, 6028, 6030, 6032,6034, 6036, 6038, 6040, 6042, 6044, 6046, 6048, 6050, 6052, 6054, 6056,6058, 6060, 6062, 6064, 6066, 6068, 6070, 6072, 6074, 6076, 6078, 6080,6082, 6084, 6086, 6088, 6090, 6092, 6094, 6096, 6098, 6100, 6102, 6104,6106, 6108, 6110, 6112, 6114, 6116, 6118, 6120, 6122, 6124, 6126, 6128,6130, 6132, 6134, 6136, 6138, 6140, 6142, 6144, 6146, 6148, 6150, 6152,6154, 6156, 6158, 6160, 6162, 6164, 6166, 6168, 6170, 6172, 6174, 6176,6178, 6180, 6182, 6184, 6186, 6188, 6190, 6192, 6194, 6196, 6198, 6200,6202, 6204, 6206, 6208, 6210, 6212, 6214, 6216, 6218, 6220, 6222, 6224,6226, 6228, 6230, 6232, 6234, 6236, 6238, 6240, 6242, 6244, 6246, 6248,6250, 6252, 6254, 6256, 6258, and 6260. In some embodiments, thepolypeptide sequence of the engineered glycosyltransferase comprises anyof SEQ ID NOS: 6016, 6018, 6020, 6022, 6024, 6026, 6028, 6030, 6032,6034, 6036, 6038, 6040, 6042, 6044, 6046, 6048, 6050, 6052, 6054, 6056,6058, 6060, 6062, 6064, 6066, 6068, 6070, 6072, 6074, 6076, 6078, 6080,6082, 6084, 6086, 6088, 6090, 6092, 6094, 6096, 6098, 6100, 6102, 6104,6106, 6108, 6110, 6112, 6114, 6116, 6118, 6120, 6122, 6124, 6126, 6128,6130, 6132, 6134, 6136, 6138, 6140, 6142, 6144, 6146, 6148, 6150, 6152,6154, 6156, 6158, 6160, 6162, 6164, 6166, 6168, 6170, 6172, 6174, 6176,6178, 6180, 6182, 6184, 6186, 6188, 6190, 6192, 6194, 6196, 6198, 6200,6202, 6204, 6206, 6208, 6210, 6212, 6214, 6216, 6218, 6220, 6222, 6224,6226, 6228, 6230, 6232, 6234, 6236, 6238, 6240, 6242, 6244, 6246, 6248,6250, 6252, 6254, 6256, 6258, and 6260.

The present invention also provides engineered glycosyltransferases,wherein the polypeptide sequences of the engineered glycosyltransferasescomprise at least one mutation or mutation set at one or more positionsselected from 14/113/158, 14/113/158/163,14/113/158/163/190/257/308/342, 14/113/158/163/437,14/113/158/308/317/330/449, 14/113/163/190/308/317,14/113/163/257/308/342/437/449, 14/113/163/257/437,14/113/163/308/317/320/322/330/449, 14/113/320/437/449, 14/158,14/158/163/190/257/308/317/330/437, 14/158/163/190/342/437,14/158/163/257, 14/158/163/257/308/449, 14/158/257/308/437/449,14/158/317/320/330/437, 14/163/317/320, 14/190, 14/190/257/317/320/322,14/257/308/320/322/330, 14/257/308/322/330/437, 14/317, 14/330, 14/449,19, 29/375, 41, 45, 46, 71, 72, 80, 81, 83, 84, 85, 88, 95, 105,113/158/163/190/257/437, 113/158/163/190/308/317/322,113/158/163/190/308/320, 113/158/163/257/308/317/322/437/449,113/158/190/257/320, 113/158/190/320/322/449, 113/158/320/322/437,113/257/308, 113/257/317/322/437, 155, 158/163,158/163/190/257/308/342/449, 158/163/308, 158/163/322/437,158/308/320/437, 158/320/437/449, 163/308/330/437/449, 168, 190/449,197, 199, 202, 209, 243, 249, 263, 273, 317/320/322/330, 322/330, 366,375, and 383, wherein the positions are numbered with reference to SEQID NO: 6138.

In some embodiments, the polypeptide sequence of the engineeredglycosyltransferase comprises at least one mutation or mutation set atone or more positions selected from 14I/113S/158P, 14I/113S/158P/163A,14I/113S/158P/163A/190R/257Q/308L/342W, 14I/113S/158P/163A/437I,14I/113S/158P/308L/317R/330D/449R, 14I/113S/163A/190R/308L/317R,14I/113S/163A/257Q/308L/342W/437I/449R, 14I/113S/163A/257Q/437I,14I/113S/163A/308L/317R/320I/322L/330D/449R, 14I/113S/320I/437I/449R,14I/158P, 14I/158P/163A/190R/257Q/308L/317R/330D/437I,14I/158P/163A/190R/342W/437I, 14I/158P/163A/257Q,14I/158P/163A/257Q/308L/449R, 14I/158P/257Q/308L/437I/449R,14I/158P/317R/320I/330D/437I, 14I/163A/317R/320I, 14I/190R,14I/190R/257Q/317R/320I/322L, 14I/257Q/308L/320I/322L/330D,14I/257Q/308L/322L/330D/437I, 14I/317R, 14I/330D, 14I/449R, 19Q,29M/375P, 41A, 45L, 46S, 71V, 725, 72T, 80P, 81T, 83A, 83K, 83N, 835,83T, 84D, 84H, 84N, 85L, 88A, 88C, 88H, 88K, 88T, 95N, 105A,113S/158P/163A/190R/257Q/437I, 113S/158P/163A/190R/308L/317R/322L,113S/158P/163A/190R/308L/320I,113S/158P/163A/257Q/308L/317R/322L/4371/449R, 1135/158P/190R/257Q/320I,113S/158P/190R/320I/322L/449R, 1135/158P/320I/322L/437I, 113S/257Q/308L,113S/257Q/317R/322L/437I, 155L, 158P/163A,158P/163A/190R/257Q/308L/342W/449R, 158P/163A/308L, 158P/163A/322L/437I,158P/308L/320/437I, 158P/320/437I/449R, 163A/308L/330D/437I/449R, 168C,168T, 190R/449R, 197K, 199E, 199H, 199M, 199Q, 199Y, 202H, 202Q, 202T,202V, 209T, 243I, 249S, 263T, 273A, 273H, 273R, 317R/320I/322L/330D,322L/330D, 366C, 366L, 366S, 366T, 366V, 375A, 375P, 375T, 375V, and383V, wherein the positions are numbered with reference to SEQ ID NO:6138. In some embodiments, the polypeptide sequence of the engineeredglycosyltransferase comprises at least one mutation or mutation set atone or more positions selected from V14I/E113S/D158P,V14I/E113S/D158P/L163A, V14I/E113S/D158P/L163A/K190R/L257Q/R308L/E342W,V14I/E113S/D158P/L163A/L437I, V14I/E113S/D158P/R308L/S317R/E330D/S449R,V14I/E113S/L163A/K190R/R308L/S317R,V14I/E113S/L163A/L257Q/R308L/E342W/L437I/S449R,V14I/E113S/L163A/L257Q/L437I,V14I/E113S/L163A/R308L/S317R/V320I/P322L/E330D/S449R,V14I/E113S/V320I/L437I/S449R, V14I/D158P,V14I/D158P/L163A/K190R/L257Q/R308L/S317R/E330D/L437I,V14I/D158P/L163A/K190R/E342W/L437I, V14I/D158P/L163A/L257Q,V14I/D158P/L163A/L257Q/R308L/S449R, V14I/D158P/L257Q/R308L/L437I/S449R,V14I/D158P/S317R/V320I/E330D/L437I, V14I/L163A/S317R/V320I, V14I/K190R,V14I/K190R/L257Q/S317R/V320I/P322L, V14I/L257Q/R308L/V320I/P322L/E330D,V14I/L257Q/R308L/P322L/E330D/L437I, V14I/S317R, V14I/E330D, V14I/S449R,V19Q, L29M/D375P, S41A, F45L, H46S, Q71V, D72S, D72T, T80P, H81T, P83A,P83K, P83N, P83S, P83T, G84D, G84H, G84N, A85L, R88A, R88C, R88H, R88K,R88T, H95N, E105A, E113S/D158P/L163A/K190R/L257Q/L437I,E113S/D158P/L163A/K190R/R308L/S317R/P322L,E113S/D158P/L163A/K190R/R308L/V320I,E113S/D158P/L163A/L257Q/R308L/S317R/P322L/L437I/S449R,E113S/D158P/K190R/L257Q/V320I, E113S/D158P/K190R/V320I/P322L/S449R,E113S/D158P/V320I/P322L/L437I, E113S/L257Q/R308L,E113S/L257Q/S317R/P322L/L437I, V155L, D158P/L163A,D158P/L163A/K190R/L257Q/R308L/E342W/S449R, D158P/L163A/R308L,D158P/L163A/P322L/L437I, D158P/R308L/V320I/L437I,D158P/V320I/L437I/S449R, L163A/R308L/E330D/L437I/S449R, P168C, P168T,K190R/S449R, Q197K, A199E, A199H, A199M, A199Q, A199Y, I202H, I202Q,I202T, I202V, E209T, L243I, Y249S, V263T, S273A, S273H, S273R,S317R/V320I/P322L/E330D, P322L/E330D, A366C, A366L, A366S, A366T, A366V,D375A, D375P, D375T, D375V, and N383V, wherein the positions arenumbered with reference to SEQ ID NO: 6138.

In some embodiments, the polypeptide sequence of the engineeredglycosyltransferase comprises a sequence at least 90% identical to anyof SEQ ID NOS: 6262, 6264, 6266, 6268, 6270, 6272, 6274, 6276, 6278,6280, 6282, 6284, 6286, 6288, 6290, 6292, 6294, 6296, 6298, 6300, 6302,6304, 6306, 6308, 6310, 6312, 6314, 6316, 6318, 6320, 6322, 6324, 6326,6328, 6330, 6332, 6334, 6336, 6338, 6340, 6342, 6344, 6346, 6348, 6350,6352, 6354, 6356, 6358, 6360, 6362, 6364, 6366, 6368, 6370, 6372, 6374,6376, 6378, 6380, 6382, 6384, 6386, 6388, 6390, 6392, 6394, 6396, 6398,6400, 6402, 6404, 6406, 6408, 6410, 6412, 6414, 6416, 6418, 6420, 6422,6424, 6426, 6428, 6430, 6432, 6434, 6436, 6438, 6440, 6442, 6444, 6446,6448, 6450, 6452, 6454, 6456, 6458, and 6460. In some embodiments, thepolypeptide sequence of the engineered glycosyltransferase comprises asequence at least 95% identical to any of SEQ ID NOS: 6262, 6264, 6266,6268, 6270, 6272, 6274, 6276, 6278, 6280, 6282, 6284, 6286, 6288, 6290,6292, 6294, 6296, 6298, 6300, 6302, 6304, 6306, 6308, 6310, 6312, 6314,6316, 6318, 6320, 6322, 6324, 6326, 6328, 6330, 6332, 6334, 6336, 6338,6340, 6342, 6344, 6346, 6348, 6350, 6352, 6354, 6356, 6358, 6360, 6362,6364, 6366, 6368, 6370, 6372, 6374, 6376, 6378, 6380, 6382, 6384, 6386,6388, 6390, 6392, 6394, 6396, 6398, 6400, 6402, 6404, 6406, 6408, 6410,6412, 6414, 6416, 6418, 6420, 6422, 6424, 6426, 6428, 6430, 6432, 6434,6436, 6438, 6440, 6442, 6444, 6446, 6448, 6450, 6452, 6454, 6456, 6458,and 6460. In some embodiments, the polypeptide sequence of theengineered glycosyltransferase comprises any of SEQ ID NOS: 6262, 6264,6266, 6268, 6270, 6272, 6274, 6276, 6278, 6280, 6282, 6284, 6286, 6288,6290, 6292, 6294, 6296, 6298, 6300, 6302, 6304, 6306, 6308, 6310, 6312,6314, 6316, 6318, 6320, 6322, 6324, 6326, 6328, 6330, 6332, 6334, 6336,6338, 6340, 6342, 6344, 6346, 6348, 6350, 6352, 6354, 6356, 6358, 6360,6362, 6364, 6366, 6368, 6370, 6372, 6374, 6376, 6378, 6380, 6382, 6384,6386, 6388, 6390, 6392, 6394, 6396, 6398, 6400, 6402, 6404, 6406, 6408,6410, 6412, 6414, 6416, 6418, 6420, 6422, 6424, 6426, 6428, 6430, 6432,6434, 6436, 6438, 6440, 6442, 6444, 6446, 6448, 6450, 6452, 6454, 6456,6458, and 6460.

The present invention also provides engineered glycosyltransferases,wherein the polypeptide sequences of the engineered glycosyltransferasescomprise at least one mutation or mutation set at one or more positionsselected from 37/71/125/174/247/248/427, 37/71/247/331/365/389/401/429,37/139/329/365/427/433/449, 37/139/427/432/433/449, 37/139/429/432/433,37/174/401/402/433/449, 37/248/331/389/427/433, 37/248/389/401/429,37/331/432/433, 71/139/389/427/429/432/433/449,71/174/329/427/429/432/433, 71/174/365/427/432, 71/248/365/389/401,71/248/449, 71/254/433/449, 71/427/432/433/449, 71/432/433,139/156/174/389/401/427/433/449, 139/156/247/365/401/433/449,139/156/247/389/401/427/433/449, 139/156/248/389,139/156/248/389/401/416/427/429/433, 139/156/365,139/174/248/331/389/401/449, 139/174/254,139/174/365/401/402/427/433/449, 139/247/248/331/401/417/432/449,139/248/254/449, 139/248/402/416/427/433/449, 139/248/432/433,139/254/401/416/427/433/449, 139/401/449, 156/248/256,156/254/331/365/427/432/449, 156/389/401/402/416/432/433,174/247/248/389/401/432/433, 174/329/432/449, 174/365,174/389/429/432/433/449, 247/248/401, 247/248/449,247/331/401/427/432/449, 247/401, 247/427/432, 248/331/427/429/433,248/365/389/427/429/432/449, 248/401/429/432/433, 248/416, 248/416/449,248/449, 254/365, 254/427/433, 331/365/429/432/433/449,365/401/402/429/432/433, 389/401/416/432/449, 389/401/427/432, 401/427,416/427/433/449, 416/432/433, 416/432/433/449, 427/432, 427/432/449,432, and 433/449, wherein the positions are numbered with reference toSEQ ID NO: 6288. In some embodiments, the polypeptide sequence of theengineered glycosyltransferase comprises at least one mutation ormutation set at one or more positions selected from37R/71L/125M/174P/247C/248W/427R, 37R/71L/247C/331K/365I/389E/401F/429W,37R/139K/329P/365I/427R/433L/449R, 37R/139K/427R/432L/433L/449R,37R/139K/429W/432L/433L, 37R/174P/401F/402L/433L/449R,37R/248W/331K/389E/427R/433L, 37R/248W/389A/401F/429W,37R/331K/432L/433L, 71L/139K/389A/427R/429W/432L/433L/449R,71L/174P/329P/427R/429W/432L/433L, 71L/174P/365I/427R/432L,71L/248W/365I/389A/401F, 71L/248W/449R, 71L/254Q/433L/449R,71L/427R/432L/433L449R, 71L/432L/433L,139K/156C/174P/389E/401F/427R/433L/449R,139K/156C/247C/365I/401F/433L/449R,139K/156C/247L/389E/401F/427R/433L/449R,139K/156C/248W/389A/401F/416S/427R/429W/433L, 139K/156C/248W/389E,139K/156C/365I, 139K/174P/248W/331K/389A/401F/449R, 139K/174P/254Q,139K/174P/365I/401F/402L/427R/433L/449R,139K/247C/248W/331K/401F/417Q/432L/449R, 139K/248W/254A/449R,139K/248W/402L/416L/427R/433L/449R, 139K/248W/432L/433L,139K/254Q/401F/416S/427R/433L/449R, 139K/401F/449R, 156C/248W/256M,156C/254Q/331K/365I/427R/432L/449R, 156C/389E/401F/402L/416L/432L/433L,174P/247C/248W/389A/401F/432L/433L, 174P/329P/432L/449R, 174P/365I,174P/389E/429W/432L/433L/449R, 247C/248W/401F,247C/331K/401F/427R/432L/449R, 247C/427R/432L, 247L/248W/449R,247L/401F, 248W/331K/427R/429W/433L, 248W/365I/389E/427R/429W/432L/449R,248W/401F/429W/432L/433L, 248W/416L, 248W/416L/449R, 248W/449R,254Q/365I, 254Q/427R/433L, 331K/365I/429W/432L/433L/449R,365I/401F/402L/429W/432L/433L, 389A/401F/416S/432L/449R,389E/401F/427R/432L, 401F/427R, 416L/427R/433L/449R,416L/432L/433L/449R, 416S/432L/433L, 427R/432L, 427R/432L/449R, 432L,and 433L/449R, wherein the positions are numbered with reference to SEQID NO: 6288. In some embodiments, the polypeptide sequence of theengineered glycosyltransferase comprises at least one mutation ormutation set at one or more positions selected fromS37R/Q71L/L125M/L174P/K247C/H248W/A427R,S37R/Q71L/K247C/R331K/V365I/D389E/L401F/L429W,S37R/P139K/G329P/V365I/A427R/A433L/S449R,S37R/P139K/A427R/K432L/A433L/S449R, S37R/P139K/L429W/K432L/A433L,S37R/L174P/L401F/E402L/A433L/S449R, S37R/H248W/R331K/D389E/A427R/A433L,S37R/H248W/D389A/L401F/L429W, S37R/R331K/K432L/A433L,Q71L/P139K/D389A/A427R/L429W/K432L/A433L/S449R,Q71L/L174P/G329P/A427R/L429W/K432L/A433L, Q71L/L174P/V365I/A427R/K432L,Q71L/H248W/V365I/D389A/L401F, Q71L/H248W/S449R, Q71L/S254Q/A433L/S449R,Q71L/A427R/K432L/A433L/S449R, Q71L/K432L/A433L,P139K/S156C/L174P/D389E/L401F/A427R/A433L/S449R,P139K/S156C/K247C/V365I/L401F/A433L/S449R,P139K/S156C/K247L/D389E/L401F/A427R/A433L/S449R,P139K/S156C/H248W/D389A/L401F/D416S/A427R/L429W/A433L,P139K/S156C/H248W/D389E, P139K/S156C/V365I,P139K/L174P/H248W/R331K/D389A/L401F/S449R, P139K/L174P/S254Q,P139K/L174P/V365I/L401F/E402L/A427R/A433L/S449R,P139K/K247C/H248W/R331K/L401F/P417Q/K432L/S449R,P139K/H248W/S254A/S449R, P139K/H248W/E402L/D416L/A427R/A433L/S449R,P139K/H248W/K432L/A433L, P139K/S254Q/L401F/D416S/A427R/A433L/S449R,P139K/L401F/S449R, S156C/H248W/L256M,S156C/S254Q/R331K/V365I/A427R/K432L/S449R,S156C/D389E/L401F/E402L/D416L/K432L/A433L,L174P/K247C/H248W/D389A/L401F/K432L/A433L, L174P/G329P/K432L/S449R,L174P/V365I, L174P/D389E/L429W/K432L/A433L/S449R, K247C/H248W/L401F,K247C/R331K/L401F/A427R/K432L/S449R, K247C/A427R/K432L,K247L/H248W/S449R, K247L/L401F, H248W/R331K/A427R/L429W/A433L,H248W/V365I/D389E/A427R/L429W/K432L/S449R,H248W/L401F/L429W/K432L/A433L, H248W/D416L, H248W/D416L/S449R,H248W/S449R, S254Q/V365I, S254Q/A427R/A433L,R331K/V365I/L429W/K432L/A433L/S449R,V365I/L401F/E402L/L429W/K432L/A433L, D389A/L401F/D416S/K432L/S449R,D389E/L401F/A427R/K432L, L401F/A427R, D416L/A427R/A433L/S449R,D416L/K432L/A433L/S449R, D416S/K432L/A433L, A427R/K432L,A427R/K432L/S449R, K432L, and A433L/S449R, wherein the positions arenumbered with reference to SEQ ID NO: 6288. In some embodiments, thepolypeptide sequence of the engineered glycosyltransferase comprises atleast one mutation or mutation set at one or more positions selectedfrom 5/449, 6, 10, 25, 25/449, 69, 69/449, 87, 87/449, 91, 91/449,144/449, 153, 153/449, 159, 159/449, 172, 172/449, 212/449, 233,233/449, 288/449, 303, 317, 347/449, 361, 369, and 421, wherein thepositions are numbered with reference to SEQ ID NO: 6288. In someembodiments, the polypeptide sequence of the engineeredglycosyltransferase comprises at least one mutation or mutation set atone or more positions selected from 5S/449R, 6P, 10K, 25I/449R,25M/449R, 25Q/449R, 25S, 69A, 69M/449R, 87A/449R, 87E, 87K, 87Q, 87R,91L, 91N/449R, 91Q, 91T/449R, 91V, 144Q/449R, 153T/449R, 153V, 159K,159R/449R, 172S, 172T/449R, 212L/449R, 233A, 233C, 233G, 233L/449R,233M/449R, 233Q/449R, 233R, 233S, 233V, 288P/449R, 303C, 303V, 317Y,347P/449R, 361C, 369K, and 421, wherein the positions are numbered withreference to SEQ ID NO: 6288. In some embodiments, the polypeptidesequence of the engineered glycosyltransferase comprises at least onemutation or mutation set at one or more positions selected fromT5S/S449R, E6P, R10K, L25I/S449R, L25M/S449R, L25Q/S449R, L25S, D69A,D69M/S449R, M87A/S449R, M87E, M87K, M87Q, M87R, I91L, I91N/S449R, I91Q,I91T/S449R, I91V, M144Q/S449R, A153T/S449R, A153V, Q159K, Q159R/S449R,H172S, H172T/S449R, A212L/S449R, W233A, W233C, W233G, W233L/S449R,W233M/S449R, W233Q/S449R, W233R, W233S, W233V, E288P/S449R, Q303C,Q303V, S317Y, K347P/S449R, T361C, V369K, and V421I, wherein thepositions are numbered with reference to SEQ ID NO: 6288. In someembodiments, the polypeptide sequence of the engineeredglycosyltransferase comprises a sequence at least 90% identical to anyof SEQ ID NOS: 6462, 6464, 6466, 6468, 6470, 6472, 6474, 6476, 6478,6480, 6482, 6484, 6486, 6488, 6490, 6492, 6494, 6496, 6498, 6500, 6502,6504, 6506, 6508, 6510, 6512, 6514, 6516, 6518, 6520, 6522, 6524, 6526,6528, 6530, 6532, 6534, 6536, 6538, 6540, 6542, 6544, 6546, 6548, 6550,6552, 6554, 6556, 6558, 6560, 6562, 6564, 6566, 6568, 6570, 6572, 6574,6576, 6578, 6580, 6582, 6584, 6586, 6588, 6590, 6592, 6594, 6596, 6598,6600, 6602, 6604, 6606, 6608, 6610, 6612, 6614, 6616, 6618, 6620, 6622,6624, 6626, 6628, 6630, 6632, 6634, 6636, 6638, 6640, 6642, 6644, 6646,6648, 6650, 6652, 6654, 6656, 6658, 6660, 6662, 6664, 6666, 6668, 6670,6672, 6674, and 6676. In some embodiments, the polypeptide sequence ofthe engineered glycosyltransferase comprises a sequence at least 95%identical to any of SEQ ID NOS: 6462, 6464, 6466, 6468, 6470, 6472,6474, 6476, 6478, 6480, 6482, 6484, 6486, 6488, 6490, 6492, 6494, 6496,6498, 6500, 6502, 6504, 6506, 6508, 6510, 6512, 6514, 6516, 6518, 6520,6522, 6524, 6526, 6528, 6530, 6532, 6534, 6536, 6538, 6540, 6542, 6544,6546, 6548, 6550, 6552, 6554, 6556, 6558, 6560, 6562, 6564, 6566, 6568,6570, 6572, 6574, 6576, 6578, 6580, 6582, 6584, 6586, 6588, 6590, 6592,6594, 6596, 6598, 6600, 6602, 6604, 6606, 6608, 6610, 6612, 6614, 6616,6618, 6620, 6622, 6624, 6626, 6628, 6630, 6632, 6634, 6636, 6638, 6640,6642, 6644, 6646, 6648, 6650, 6652, 6654, 6656, 6658, 6660, 6662, 6664,6666, 6668, 6670, 6672, 6674, and 6676. In some embodiments, thepolypeptide sequence of the engineered glycosyltransferase comprises anyof SEQ ID NOS: 6462, 6464, 6466, 6468, 6470, 6472, 6474, 6476, 6478,6480, 6482, 6484, 6486, 6488, 6490, 6492, 6494, 6496, 6498, 6500, 6502,6504, 6506, 6508, 6510, 6512, 6514, 6516, 6518, 6520, 6522, 6524, 6526,6528, 6530, 6532, 6534, 6536, 6538, 6540, 6542, 6544, 6546, 6548, 6550,6552, 6554, 6556, 6558, 6560, 6562, 6564, 6566, 6568, 6570, 6572, 6574,6576, 6578, 6580, 6582, 6584, 6586, 6588, 6590, 6592, 6594, 6596, 6598,6600, 6602, 6604, 6606, 6608, 6610, 6612, 6614, 6616, 6618, 6620, 6622,6624, 6626, 6628, 6630, 6632, 6634, 6636, 6638, 6640, 6642, 6644, 6646,6648, 6650, 6652, 6654, 6656, 6658, 6660, 6662, 6664, 6666, 6668, 6670,6672, 6674, and 6676.

The present invention also provides engineered glycosyltransferases,wherein the polypeptide sequences of the engineered glycosyltransferasescomprise at least one mutation or mutation set at one or more positionsselected from 37/41/45/72/81/83/84/88/155/163/168/190/331/375,37/41/45/72/81/83/84/88/155/263/273/331/375/432,37/41/45/72/81/83/84/88/155/331/366/375,37/41/45/72/81/83/84/88/155/331/375, 37/41/45/72/81/83/84/88/248/375,37/41/45/72/81/83/84/88/331/366/432/433,37/41/45/72/88/155/168/190/331/366/375/432,37/41/45/72/88/155/168/190/331/375, 37/41/45/72/88/190/263/273/331/366,37/41/45/72/155/163/168/190/243/263/273/331/366/432,37/41/45/72/155/163/168/243/248/273/331/366/432/433,37/41/45/72/155/163/168/263/366/432/433,37/41/45/72/155/163/331/366/375,37/41/45/72/155/190/243/248/273/331/432/433,37/41/45/72/155/190/248/263/331, 37/41/45/72/155/190/263/331/366,37/41/45/72/155/190/273/331/366/375, 37/41/45/72/155/190/273/366,37/41/45/72/155/190/273/366/375/432, 37/41/45/72/155/248/263/273/366,37/41/45/72/155/263/331/375, 37/41/45/72/155/263/375,37/41/45/72/155/273/366, 37/41/45/72/155/331/366/375/432/433,37/41/45/72/190/263/273/331, 37/41/45/72/190/331/366,37/41/45/72/190/366, 37/41/45/72/263/331/366,37/41/45/72/263/366/432/433, 37/41/45/72/331/366,37/41/45/81/83/84/88/155/168/190/243/331/366,37/41/45/81/83/84/88/155/168/331/375,37/41/45/81/83/84/88/155/263/273/331/366,37/41/45/81/83/84/88/163/168/263/273/331/366/375,37/41/45/155/163/168/263/331/375, 37/41/45/155/168/248/273/331/375,37/41/45/155/190/331/366/375, 37/41/45/155/366,37/41/45/155/366/432/433, 37/41/45/155/375, 37/41/45/243/248/273/331,37/41/45/263/331/375/432, 37/41/45/331/366/432/433,37/72/81/83/88/155/190, 37/72/197/273/331/375/432,37/83/263/365/366/375, 37/190/202, 41/45/72/155/263/331/366/375/432/433,41/45/163/168/243/248/273/366/432, 45/72/84/88/197/375, 45/72/88/366,45/72/163/202/365/366/375, 45/72/168/243/331/365/366/429/432,45/84/168/190/199/254/273/365/366, 45/163/168/190/199/366/429/432,45/163/168/197/263/331/365/366, 72/81/83/84/88/155/163/168/190/366,72/81/83/84/88/155/190, 72/81/83/84/88/155/190/273/331/366/432,72/81/83/84/88/155/273/331/375, 72/81/83/84/88/155/366,72/81/83/84/88/163/168/190/243/263/331/366,72/81/83/84/88/163/168/263/331/375, 72/81/84/190/248,72/83/84/88/202/254/273/366/375, 72/83/84/197/202/243/263/365/366,72/83/88/243/263/331/365/366, 72/155/163/168/190/366/432/433,72/155/190, 72/155/190/263/331/366, 72/155/190/366,72/155/273/331/375/432, 72/243/248/263/366/432/433,72/243/248/273/366/432/433, 72/243/248/366/432/433, 72/248,81/83/84/88/155/163/168/273/331/375, 81/83/84/88/155/190/263/331/366,81/83/84/88/155/263/366/375, 81/83/84/88/155/366,81/83/84/88/190/263/375, 81/83/84/88/263/273/331/366/432,81/83/169/190/263, 81/83/190/263/365/366, 81/83/202/365/366/402,81/88/375/402, 83/88/155/273/366/375,84/155/168/197/199/331/366/375/383/402, 84/168/197/202/263/366,84/197/366/402, 155/168/190/197/199/366, 155/168/375,155/263/366/432/433, 190/199/202/331/366, 197/199/202, 197/202/248,197/248, 199/263/331/365/366, 248/375, 365/366, and 365/375/402, whereinthe positions are numbered with reference to SEQ ID NO: 6468. In someembodiments, the polypeptide sequence of the engineeredglycosyltransferase comprises at least one mutation or mutation set atone or more positions selected from37R/41A/45L/72T/81T/83S/84N/88T/155L/163A/168T/190R/331K/375P,37R/41A/45L/72T/81T/83S/84N/88T/155L/263T/273R/331K/375P/432L,37R/41A/45L/72T/81T/83S/84N/88T/155L/331K/366C/375P,37R/41A/45L/72T/81T/83S/84N/88T/155L/331K/375P,37R/41A/45L/72T/81T/83S/84N/88T/248W/375P,37R/41A/45L/72T/81T/83S/84N/88T/331K/366V/432L/433A,37R/41A/45L/72T/88T/155L/168T/190R/331K/366C/375P/432L,37R/41A/45L/72T/88T/155L/168T/190R/331K/375P,37R/41A/45L/72T/88T/190R/263T/273R/331K/366C,37R/41A/45L/72T/155L/163A/168T/190R/243I/263T/273R/331K/366C/432L,37R/41A/45L/72T/155L/163A/168T/243I/248W/273R/331K/366V/432L/433A,37R/41A/45L/72T/155L/163A/168T/263T/366C/432L/433A,37R/41A/45L/72T/155L/163A/331K/366C/375P,37R/41A/45L/72T/155L/190R/243I/248W/273R/331K/432L/433A,37R/41A/45L/72T/155L/190R/248W/263T/331K,37R/41A/45L/72T/155L/190R/263T/331K/366C,37R/41A/45L/72T/155L/190R/273R/331K/366V/375P,37R/41A/45L/72T/155L/190R/273R/366C,37R/41A/45L/72T/155L/190R/273R/366C/375P/432L,37R/41A/45L/72T/155L/248W/263T/273R/366C,37R/41A/45L/72T/155L/263T/331K/375P, 37R/41A/45L/72T/155L/263T/375P,37R/41A/45L/72T/155L/273R/366C,37R/41A/45L/72T/155L/331K/366V/375P/432L/433A,37R/41A/45L/72T/190R/263T/273R/331K, 37R/41A/45L/72T/190R/331K/366C,37R/41A/45L/72T/190R/366C, 37R/41A/45L/72T/263T/331K/366C,37R/41A/45L/72T/263T/366C/432L/433A, 37R/41A/45L/72T/331K/366C,37R/41A/45L/81T/83S/84N/88T/155L/168T/190R/243I/331K/366C,37R/41A/45L/81T/83S/84N/88T/155L/168T/331K/375P,37R/41A/45L/81T/83S/84N/88T/155L/263T/273R/331K/366C,37R/41A/45L/81T/83S/84N/88T/163A/168T/263T/273R/331K/366C/375P,37R/41A/45L/155L/163A/168T/263T/331K/375P,37R/41A/45L/155L/168T/248W/273R/331K/375P,37R/41A/45L/155L/190R/331K/366V/375P, 37R/41A/45L/155L/366C,37R/41A/45L/155L/366C/432L/433A, 37R/41A/45L/155L/375P,37R/41A/45L/243I/248W/273R/331K, 37R/41A/45L/263T/331K/375P/432L,37R/41A/45L/331K/366V/432L/433A, 37R/72T/81T/83S/88T/155L/190R,37R/72T/197K/273R/331K/375P/432L, 37R/83S/263T/365I/366V/375P,37R/190R/202H, 41A/45L/72T/155L/263T/331K/366V/375P/432L/433A,41A/45L/163A/168T/243I/248W/273R/366C/432L, 45L/72T/84N/88T/197K/375P,45L/72T/88T/366C, 45L/72T/163A/202H/365I/366V/375P,45L/72T/168T/243I/331K/365I/366C/429W/432L,45L/84N/168T/190R/199Q/254A/273R/365I/366C,45L/163A/168T/190R/199Q/366C/429W/432L,45L/163A/168T/197K/263T/331K/365I/366C,72T/81T/83S/84N/88T/155L/163A/168T/190R/366C,72T/81T/83S/84N/88T/155L/190R,72T/81T/83S/84N/88T/155L/190R/273R/331K/366V/432L,72T/81T/83S/84N/88T/155L/273R/331K/375P, 72T/81T/83S/84N/88T/155L/366C,72T/81T/83S/84N/88T/163A/168T/190R/243I/263T/331K/366C,72T/81T/83S/84N/88T/163A/168T/263T/331K/375P, 72T/81T/84N/190R/248W,72T/83S/84N/88T/202H/254A/273R/366C/375P,72T/83S/84N/197K/202H/243I/263T/365I/366C,72T/83S/88T/243I/263T/331K/365I/366C,72T/155L/163A/168T/190R/366V/432L/433A, 72T/155L/190R,72T/155L/190R/263T/331K/366C, 72T/155L/190R/366C,72T/155L/273R/331K/375P/432L, 72T/243I/248W/263T/366V/432L/433A,72T/243I/248W/273R/366V/432L/433A, 72T/243I/248W/366V/432L/433A,72T/248W, 81T/83S/84N/88T/155L/163A/168T/273R/331K/375P,81T/83S/84N/88T/155L/190R/263T/331K/366C,81T/83S/84N/88T/155L/263T/366V/375P, 81T/83S/84N/88T/155L/366C,81T/83S/84N/88T/190R/263T/375P,81T/83S/84N/88T/263T/273R/331K/366C/432L, 81T/83S/169D/190R/263T,81T/83S/190R/263T/365I/366C, 81T/83S/202H/365I/366C/402L,81T/88T/375P/402L, 83S/88T/155L/273R/366V/375P,84N/155L/168T/197K/199Q/331K/366V/375P/383V/402L,84N/168T/197K/202H/263T/366C, 84N/197K/366C/402L,155L/168T/190R/197K/199Q/366C, 155L/168T/375P, 155L/263T/366C/432L/433A,190R/199Q/202H/331K/366C, 197K/199Q/202H, 197K/202H/248W, 197K/248W,199Q/263T/331K/365I/366C, 248W/375P, 365I/366C, and 365I/375P/402L,wherein the positions are numbered with reference to SEQ ID NO: 6468. Insome embodiments, the polypeptide sequence of the engineeredglycosyltransferase comprises at least one mutation or mutation set atone or more positions selected fromS37R/S41A/F45L/D72T/H81T/P83S/G84N/R88T/V155L/L163A/P168T/K190R/R331K/D375P,S37R/S41A/F45L/D72T/H81T/P83S/G84N/R88T/V155L/V263T/S273R/R331K/D375P/K432L,S37R/S41A/F45L/D72T/H81T/P83S/G84N/R88T/V155L/R331K/A366C/D375P,S37R/S41A/F45L/D72T/H81T/P83S/G84N/R88T/V155L/R331K/D375P,S37R/S41A/F45L/D72T/H81T/P83S/G84N/R88T/H248W/D375P,S37R/S41A/F45L/D72T/H81T/P83S/G84N/R88T/R331K/A366V/K432L/L433A,S37R/S41A/F45L/D72T/R88T/V155L/P168T/K190R/R331K/A366C/D375P/K432L,S37R/S41A/F45L/D72T/R88T/V155L/P168T/K190R/R331K/D375P,S37R/S41A/F45L/D72T/R88T/K190R/V263T/S273R/R331K/A366C,S37R/S41A/F45L/D72T/V155L/L163A/P168T/K190R/L243I/V263T/S273R/R331K/A366C/K432L,S37R/S41A/F45L/D72T/V155L/L163A/P168T/L243I/H248W/S273R/R331K/A366V/K432L/L433A,S37R/S41A/F45L/D72T/V155L/L163A/P168T/V263T/A366C/K432L/L433A,S37R/S41A/F45L/D72T/V155L/L163A/R331K/A366C/D375P,S37R/S41A/F45L/D72T/V155L/K190R/L243I/H248W/S273R/R331K/K432L/L433A,S37R/S41A/F45L/D72T/V155L/K190R/H248W/V263T/R331K,S37R/S41A/F45L/D72T/V155L/K190R/V263T/R331K/A366C,S37R/S41A/F45L/D72T/V155L/K190R/S273R/R331K/A366V/D375P,S37R/S41A/F45L/D72T/V155L/K190R/S273R/A366C,S37R/S41A/F45L/D72T/V155L/K190R/S273R/A366C/D375P/K432L,S37R/S41A/F45L/D72T/V155L/H248W/V263T/S273R/A366C,S37R/S41A/F45L/D72T/V155L/V263T/R331K/D375P,S37R/S41A/F45L/D72T/V155L/V263T/D375P,S37R/S41A/F45L/D72T/V155L/S273R/A366C,S37R/S41A/F45L/D72T/V155L/R331K/A366V/D375P/K432L/L433A,S37R/S41A/F45L/D72T/K190R/V263T/S273R/R331K,S37R/S41A/F45L/D72T/K190R/R331K/A366C, S37R/S41A/F45L/D72T/K190R/A366C,S37R/S41A/F45L/D72T/V263T/R331K/A366C,S37R/S41A/F45L/D72T/V263T/A366C/K432L/L433A,S37R/S41A/F45L/D72T/R331K/A366C,S37R/S41A/F45L/H81T/P83S/G84N/R88T/V155L/P168T/K190R/L243I/R331K/A366C,S37R/S41A/F45L/H81T/P83S/G84N/R88T/V155L/P168T/R331K/D375P,S37R/S41A/F45L/H81T/P83S/G84N/R88T/V155L/V263T/S273R/R331K/A366C,S37R/S41A/F45L/H81T/P83S/G84N/R88T/L163A/P168T/V263T/S273R/R331K/A366C/D375P,S37R/S41A/F45L/V155L/L163A/P168T/V263T/R331K/D375P,S37R/S41A/F45L/V155L/P168T/H248W/S273R/R331K/D375P,S37R/S41A/F45L/V155L/K190R/R331K/A366V/D375P,S37R/S41A/F45L/V155L/A366C, S37R/S41A/F45L/V155L/A366C/K432L/L433A,S37R/S41A/F45L/V155L/D375P, S37R/S41A/F45L/L243I/H248W/S273R/R331K,S37R/S41A/F45L/V263T/R331K/D375P/K432L,S37R/S41A/F45L/R331K/A366V/K432L/L433A,S37R/D72T/H81T/P83S/R88T/V155L/K190R,S37R/D72T/Q197K/S273R/R331K/D375P/K432L,S37R/P83S/V263T/V365I/A366V/D375P, S37R/K190R/I202H,S41A/F45L/D72T/V155L/V263T/R331K/A366V/D375P/K432L/L433A,S41A/F45L/L163A/P168T/L243I/H248W/S273R/A366C/K432L,F45L/D72T/G84N/R88T/Q197K/D375P, F45L/D72T/R88T/A366C,F45L/D72T/L163A/I202H/V365I/A366V/D375P,F45L/D72T/P168T/L243I/R331K/V365I/A366C/L429W/K432L,F45L/G84N/P168T/K190R/A199Q/S254A/S273R/V365I/A366C,F45L/L163A/P168T/K190R/A199Q/A366C/L429W/K432L,F45L/L163A/P168T/Q197K/V263T/R331K/V365I/A366C,D72T/H81T/P83S/G84N/R88T/V155L/L163A/P168T/K190R/A366C,D72T/H81T/P83S/G84N/R88T/V155L/K190R,D72T/H81T/P83S/G84N/R88T/V155L/K190R/S273R/R331K/A366V/K432L,D72T/H81T/P83S/G84N/R88T/V155L/S273R/R331K/D375P,D72T/H81T/P83S/G84N/R88T/V155L/A366C,D72T/H81T/P83S/G84N/R88T/L163A/P168T/K190R/L243I/V263T/R331K/A366C,D72T/H81T/P83S/G84N/R88T/L163A/P168T/V263T/R331K/D375P,D72T/H81T/G84N/K190R/H248W,D72T/P83S/G84N/R88T/I202H/S254A/S273R/A366C/D375P,D72T/P83S/G84N/Q197K/I202H/L243I/V263T/V365I/A366C,D72T/P83S/R88T/L243I/V263T/R331K/V365I/A366C,D72T/V155L/L163A/P168T/K190R/A366V/K432L/L433A, D72T/V155L/K190R,D72T/V155L/K190R/V263T/R331K/A366C, D72T/V155L/K190R/A366C,D72T/V155L/S273R/R331K/D375P/K432L,D72T/L243I/H248W/V263T/A366V/K432L/L433A,D72T/L243I/H248W/S273R/A366V/K432L/L433A,D72T/L243I/H248W/A366V/K432L/L433A, D72T/H248W,H81T/P83S/G84N/R88T/V155L/L163A/P168T/S273R/R331K/D375P,H81T/P83S/G84N/R88T/V155L/K190R/V263T/R331K/A366C,H81T/P83S/G84N/R88T/V155L/V263T/A366V/D375P,H81T/P83S/G84N/R88T/V155L/A366C, H81T/P83S/G84N/R88T/K190R/V263T/D375P,H81T/P83S/G84N/R88T/V263T/S273R/R331K/A366C/K432L,H81T/P83S/E169D/K190R/V263T, H81T/P83S/K190R/V263T/V365I/A366C,H81T/P83S/I202H/V365I/A366C/E402L, H81T/R88T/D375P/E402L,P83S/R88T/V155L/S273R/A366V/D375P,G84N/V155L/P168T/Q197K/A199Q/R331K/A366V/D375P/N383V/E402L,G84N/P168T/Q197K/I202H/V263T/A366C, G84N/Q197K/A366C/E402L,V155L/P168T/K190R/Q197K/A199Q/A366C, V155L/P168T/D375P,V155L/V263T/A366C/K432L/L433A, K190R/A199Q/I202H/R331K/A366C,Q197K/A199Q/I202H, Q197K/I202H/H248W, Q197K/H248W,A199Q/V263T/R331K/V365I/A366C, H248W/D375P, V365I/A366C, andV365I/D375P/E402L, wherein the positions are numbered with reference toSEQ ID NO: 6468. In some embodiments, the polypeptide sequence of theengineered glycosyltransferase comprises at least one mutation ormutation set at one or more positions selected from 2, 3, 4, 9, 53/437,61, 64, 72/170, 72/405, 94, 96, 98, 113, 118, 118/120, 120, 129,134/158, 158, 165, 170, 171, 173, 183, 193, 214, 214/222, 222, 226, 229,234, 253, 265, 269, 272, 289, 296, 300, 302, 304, 322, 322/407, 330,390, 395/439, 396, 398, 399, 403, 405, 408, 411, 412, 423, 428, 434,435, 438, 439, 442, 444, 448, 449, 452, and 454, wherein the positionsare numbered with reference to SEQ ID NO: 6468. In some embodiments, thepolypeptide sequence of the engineered glycosyltransferase comprises atleast one mutation or mutation set at one or more positions selectedfrom 2F, 2H, 2K, 2M, 2Q, 2R, 2V, 3F, 3L, 3M, 4N, 4S, 9A, 9G, 9K, 9M, 9S,53E/437T, 61Q, 64L, 64M, 72E/170A, 72E/405S, 94R, 96C, 96K, 98E, 985,98T, 113D, 113G, 113P, 118C, 118T/120V, 118V, 120V, 129P, 134E/158N,158T, 165L, 170A, 170G, 170H, 170P, 170V, 171A, 171L, 171P, 171Q, 173I,173K, 173L, 173S, 183I, 183L, 183P, 193F, 214K, 214R, 214R/222H, 222A,222N, 222Q, 222R, 226S, 229M, 229Q, 234N, 2345, 234T, 253D, 253E, 253N,253P, 253T, 253V, 265H, 269L, 269M, 269N, 269R, 2725, 289D, 289G, 289N,289R, 296Q, 300A, 300E, 302G, 304K, 304P, 322A, 322G, 322K, 322P,322P/407I, 3225, 322T, 322V/407I, 330S, 390I, 390R, 395I/439V, 396T,396V, 3985, 399P, 399Q, 3995, 403V, 405A, 405D, 405P, 405S, 405T, 408D,408K, 408S, 411H, 411K, 411T, 412K, 423T, 428E, 428G, 428I, 428L, 428N,428Q, 428R, 4285, 428T, 428V, 428Y, 434E, 434G, 435A, 435D, 435E, 435G,435I, 435K, 435L, 435N, 435S, 435V, 435Y, 438E, 438R, 438S, 439A, 439E,439G, 439H, 439M, 439R, 439W, 442F, 442T, 444A, 448K, 448Q, 449G, 449L,449S, 452T, and 454V, wherein the positions are numbered with referenceto SEQ ID NO: 6468. In some embodiments, the polypeptide sequence of theengineered glycosyltransferase comprises at least one mutation ormutation set at one or more positions selected from P2F, P2H, P2K, P2M,P2Q, P2R, P2V, N3F, N3L, N3M, T4N, T4S, V9A, V9G, V9K, V9M, V9S,K53E/I437T, S61Q, F64L, F64M, D72E/D170A, D72E/E405S, K94R, G96C, G96K,D98E, D98S, D98T, S113D, S113G, S113P, A118C, A118T/L120V, A118V, L120V,A129P, D134E/P158N, P158T, Y165L, D170A, D170G, D170H, D170P, D170V,K171A, K171L, K171P, K171Q, R173I, R173K, R173L, R173S, M183I, M183L,M183P, Y193F, S214K, S214R, S214R/K222H, K222A, K222N, K222Q, K222R,E226S, L229M, L229Q, R234N, R234S, R234T, S253D, S253E, S253N, S253P,S253T, S253V, Q265H, Q269L, Q269M, Q269N, Q269R, P272S, K289D, K289G,K289N, K289R, R296Q, D300A, D300E, K302G, S304K, S304P, L322A, L322G,L322K, L322P, L322P/V407I, L322S, L322T, L322V/V407I, E330S, V390I,V390R, V395I/P439V, Y396T, Y396V, E398S, N399P, N399Q, N399S, R403V,E405A, E405D, E405P, E405S, E405T, N408D, N408K, N408S, R411H, R411K,R411T, R412K, R423T, K428E, K428G, K428I, K428L, K428N, K428Q, K428R,K428S, K428T, K428V, K428Y, D434E, D434G, R435A, R435D, R435E, R435G,R435I, R435K, R435L, R435N, R435S, R435V, R435Y, A438E, A438R, A438S,P439A, P439E, P439G, P439H, P439M, P439R, P439W, S442F, S442T, Y444A,E448K, E448Q, R449G, R449L, R449S, S452T, and I454V, wherein thepositions are numbered with reference to SEQ ID NO: 6468. In someembodiments, the polypeptide sequence of the engineeredglycosyltransferase comprises a sequence at least 90% identical to anyof SEQ ID NOS: 6678, 6680, 6682, 6684, 6686, 6688, 6690, 6692, 6694,6696, 6698, 6700, 6702, 6704, 6706, 6708, 6710, 6712, 6714, 6716, 6718,6720, 6722, 6724, 6726, 6728, 6730, 6732, 6734, 6736, 6738, 6740, 6742,6744, 6746, 6748, 6750, 6752, 6754, 6756, 6758, 6760, 6762, 6764, 6766,6768, 6770, 6772, 6774, 6776, 6778, 6780, 6782, 6784, 6786, 6788, 6790,6792, 6794, 6796, 6798, 6800, 6802, 6804, 6806, 6808, 6810, 6812, 6814,6816, 6818, 6820, 6822, 6824, 6826, 6828, 6830, 6832, 6834, 6836, 6838,6840, 6842, 6844, 6846, 6848, 6850, 6852, 6854, 6856, 6858, 6860, 6862,6864, 6866, 6868, 6870, 6872, 6874, 6876, 6878, 6880, 6882, 6884, 6886,6888, 6890, 6892, 6894, 6896, 6898, 6900, 6902, 6904, 6906, 6908, 6910,6912, 6914, 6916, 6918, 6920, 6922, 6924, 6926, 6928, 6930, 6932, 6934,6936, 6938, 6940, 6942, 6944, 6946, 6948, 6950, 6952, 6954, 6956, 6958,6960, 6962, 6964, 6966, 6968, 6970, 6972, 6974, 6976, 6978, 6980, 6982,6984, 6986, 6988, 6990, 6992, 6994, 6996, 6998, 7000, 7002, 7004, 7006,7008, 7010, 7012, 7014, 7016, 7018, 7020, 7022, 7024, 7026, 7028, 7030,7032, 7034, 7036, 7038, 7040, 7042, 7044, 7046, 7048, 7050, 7052, 7054,7056, 7058, 7060, 7062, 7064, 7066, 7068, 7070, 7072, 7074, 7076, 7078,7080, 7082, 7084, 7086, 7088, 7090, 7092, 7094, 7096, 7098, 7100, 7102,7104, 7106, 7108, 7110, 7112, 7114, 7116, 7118, 7120, 7122, 7124, 7126,7128, 7130, 7132, 7134, 7136, 7138, 7140, 7142, 7144, 7146, 7148, 7150,7152, 7154, 7156, 7158, 7160, 7162, 7164, 7166, 7168, 7170, 7172, 7174,7176, 7178, 7180, 7182, 7184, 7186, 7188, 7190, 7192, 7194, 7196, 7198,7200, 7202, 7204, 7206, 7208, 7210, 7212, and 7214. In some embodiments,the polypeptide sequence of the engineered glycosyltransferase comprisesa sequence at least 95% identical to any of SEQ ID NOS: 6678, 6680,6682, 6684, 6686, 6688, 6690, 6692, 6694, 6696, 6698, 6700, 6702, 6704,6706, 6708, 6710, 6712, 6714, 6716, 6718, 6720, 6722, 6724, 6726, 6728,6730, 6732, 6734, 6736, 6738, 6740, 6742, 6744, 6746, 6748, 6750, 6752,6754, 6756, 6758, 6760, 6762, 6764, 6766, 6768, 6770, 6772, 6774, 6776,6778, 6780, 6782, 6784, 6786, 6788, 6790, 6792, 6794, 6796, 6798, 6800,6802, 6804, 6806, 6808, 6810, 6812, 6814, 6816, 6818, 6820, 6822, 6824,6826, 6828, 6830, 6832, 6834, 6836, 6838, 6840, 6842, 6844, 6846, 6848,6850, 6852, 6854, 6856, 6858, 6860, 6862, 6864, 6866, 6868, 6870, 6872,6874, 6876, 6878, 6880, 6882, 6884, 6886, 6888, 6890, 6892, 6894, 6896,6898, 6900, 6902, 6904, 6906, 6908, 6910, 6912, 6914, 6916, 6918, 6920,6922, 6924, 6926, 6928, 6930, 6932, 6934, 6936, 6938, 6940, 6942, 6944,6946, 6948, 6950, 6952, 6954, 6956, 6958, 6960, 6962, 6964, 6966, 6968,6970, 6972, 6974, 6976, 6978, 6980, 6982, 6984, 6986, 6988, 6990, 6992,6994, 6996, 6998, 7000, 7002, 7004, 7006, 7008, 7010, 7012, 7014, 7016,7018, 7020, 7022, 7024, 7026, 7028, 7030, 7032, 7034, 7036, 7038, 7040,7042, 7044, 7046, 7048, 7050, 7052, 7054, 7056, 7058, 7060, 7062, 7064,7066, 7068, 7070, 7072, 7074, 7076, 7078, 7080, 7082, 7084, 7086, 7088,7090, 7092, 7094, 7096, 7098, 7100, 7102, 7104, 7106, 7108, 7110, 7112,7114, 7116, 7118, 7120, 7122, 7124, 7126, 7128, 7130, 7132, 7134, 7136,7138, 7140, 7142, 7144, 7146, 7148, 7150, 7152, 7154, 7156, 7158, 7160,7162, 7164, 7166, 7168, 7170, 7172, 7174, 7176, 7178, 7180, 7182, 7184,7186, 7188, 7190, 7192, 7194, 7196, 7198, 7200, 7202, 7204, 7206, 7208,7210, 7212, and 7214. In some embodiments, the polypeptide sequence ofthe engineered glycosyltransferase comprises any of SEQ ID NOS: 6678,6680, 6682, 6684, 6686, 6688, 6690, 6692, 6694, 6696, 6698, 6700, 6702,6704, 6706, 6708, 6710, 6712, 6714, 6716, 6718, 6720, 6722, 6724, 6726,6728, 6730, 6732, 6734, 6736, 6738, 6740, 6742, 6744, 6746, 6748, 6750,6752, 6754, 6756, 6758, 6760, 6762, 6764, 6766, 6768, 6770, 6772, 6774,6776, 6778, 6780, 6782, 6784, 6786, 6788, 6790, 6792, 6794, 6796, 6798,6800, 6802, 6804, 6806, 6808, 6810, 6812, 6814, 6816, 6818, 6820, 6822,6824, 6826, 6828, 6830, 6832, 6834, 6836, 6838, 6840, 6842, 6844, 6846,6848, 6850, 6852, 6854, 6856, 6858, 6860, 6862, 6864, 6866, 6868, 6870,6872, 6874, 6876, 6878, 6880, 6882, 6884, 6886, 6888, 6890, 6892, 6894,6896, 6898, 6900, 6902, 6904, 6906, 6908, 6910, 6912, 6914, 6916, 6918,6920, 6922, 6924, 6926, 6928, 6930, 6932, 6934, 6936, 6938, 6940, 6942,6944, 6946, 6948, 6950, 6952, 6954, 6956, 6958, 6960, 6962, 6964, 6966,6968, 6970, 6972, 6974, 6976, 6978, 6980, 6982, 6984, 6986, 6988, 6990,6992, 6994, 6996, 6998, 7000, 7002, 7004, 7006, 7008, 7010, 7012, 7014,7016, 7018, 7020, 7022, 7024, 7026, 7028, 7030, 7032, 7034, 7036, 7038,7040, 7042, 7044, 7046, 7048, 7050, 7052, 7054, 7056, 7058, 7060, 7062,7064, 7066, 7068, 7070, 7072, 7074, 7076, 7078, 7080, 7082, 7084, 7086,7088, 7090, 7092, 7094, 7096, 7098, 7100, 7102, 7104, 7106, 7108, 7110,7112, 7114, 7116, 7118, 7120, 7122, 7124, 7126, 7128, 7130, 7132, 7134,7136, 7138, 7140, 7142, 7144, 7146, 7148, 7150, 7152, 7154, 7156, 7158,7160, 7162, 7164, 7166, 7168, 7170, 7172, 7174, 7176, 7178, 7180, 7182,7184, 7186, 7188, 7190, 7192, 7194, 7196, 7198, 7200, 7202, 7204, 7206,7208, 7210, 7212, and 7214.

The present invention also provides engineered glycosyltransferases,wherein the polypeptide sequences of the engineered glycosyltransferasescomprise at least one mutation or mutation set at one or more positionsselected from 5/6/25/317, 5/6/69/288/303, 5/6/91/288/317,5/25/91/212/303/317, 5/25/91/263, 5/25/288, 5/69/91/212/288,5/69/91/212/303, 5/91/288/303, 5/91/303, 5/91/317/421, 5/288,6/91/212/288/303/369/421, 6/212/288/303, 6/288, 25/91/212/288,25/91/263/288/303, 25/91/303/317/369, 25/91/317/369, 25/263/317, 87/144,87/144/159/361, 87/144/159/361/433, 87/433, 88/144, 88/144/159,91/263/317/369, 91/288/303/317/369, 91/288/317/369/421, 91/303, 91/317,91/317/369, 109/144/153/155/159/433, 144, 212/288, 263/288/303/317, and288, wherein the positions are numbered with reference to SEQ ID NO:6864. In some embodiments, the polypeptide sequence of the engineeredglycosyltransferase comprises at least one mutation or mutation set atone or more positions selected from 5S/6P/25I/317Y, 5S/6P/69A/288P/303V,5S/6P/91Q/288P/317Y, 5S/25Q/91Q/212L/303V/317Y, 5S/25Q/91Q/263T,5S/25Q/288P, 5S/69A/91Q/212L/288P, 5S/69A/91Q/212L/303V,5S/91Q/288P/303V, 5S/91Q/303V, 5S/91Q/317Y/421I, 5S/288P,6P/91Q/212L/288P/303V/369K/421I, 6P/212L/288P/303V, 6P/288P,25Q/91Q/212L/288P, 25Q/91Q/263T/288P/303V, 25Q/91Q/317Y/369K,25Q/91T/303V/317Y/369K, 25Q/263T/317Y, 87K/144Q, 87K/144Q/159K/361C,87K/144Q/159K/361C/433A, 87K/433A, 88T/144Q, 88T/144Q/159K,91Q/263T/317Y/369K, 91Q/288P/303V/317Y/369K, 91Q/288P/317Y/369K/421I,91Q/303V, 91Q/317Y, 91Q/317Y/369K, 109W/144Q/153V/155L/159K/433A, 144Q,212L/288P, 263T/288P/303V/317Y, and 288P, wherein the positions arenumbered with reference to SEQ ID NO: 6864. In some embodiments, thepolypeptide sequence of the engineered glycosyltransferase comprises atleast one mutation or mutation set at one or more positions selectedfrom T5S/E6P/L25I/S317Y, T5S/E6P/D69A/E288P/Q303V,T5S/E6P/I91Q/E288P/S317Y, T5S/L25Q/I91Q/A212L/Q303V/S317Y,T5S/L25Q/I91Q/V263T, T5S/L25Q/E288P, T5S/D69A/I91Q/A212L/E288P,T5S/D69A/I91Q/A212L/Q303V, T5S/I91Q/E288P/Q303V, T5S/I91Q/Q303V,T5S/I91Q/S317Y/V421I, T5S/E288P, E6P/I91Q/A212L/E288P/Q303V/V369K/V421I,E6P/A212L/E288P/Q303V, E6P/E288P, L25Q/I91Q/A212L/E288P,L25Q/I91Q/V263T/E288P/Q303V, L25Q/I91Q/S317Y/V369K,L25Q/I91T/Q303V/S317Y/V369K, L25Q/V263T/S317Y, M87K/M144Q,M87K/M144Q/Q159K/T361C, M87K/M144Q/Q159K/T361C/L433A, M87K/L433A,R88T/M144Q, R88T/M144Q/Q159K, I91Q/V263T/S317Y/V369K,I91Q/E288P/Q303V/S317Y/V369K, I91Q/E288P/S317Y/V369K/V421I, I91Q/Q303V,I91Q/S317Y, I91Q/S317Y/V369K, R109W/M144Q/A153V/V155L/Q159K/L433A,M144Q, A212L/E288P, V263T/E288P/Q303V/S317Y, and E288P, wherein thepositions are numbered with reference to SEQ ID NO: 6864. In someembodiments, the polypeptide sequence of the engineeredglycosyltransferase comprises a sequence at least 90% identical to anyof SEQ ID NOS: 7362, 7364, 7366, 7368, 7370, 7372, 7374, 7376, 7378,7380, 7382, 7384, 7386, 7388, 7390, 7392, 7394, 7396, 7398, 7400, 7402,7404, 7406, 7408, 7410, 7412, 7414, 7416, 7418, 7420, 7422, 7424, 7426,7428, 7430, 7432, 7434, and 7436, wherein the positions are numberedwith reference to SEQ ID NO: 6864. In some embodiments, the polypeptidesequence of the engineered glycosyltransferase comprises a sequence atleast 95% identical to any of SEQ ID NOS: 7362, 7364, 7366, 7368, 7370,7372, 7374, 7376, 7378, 7380, 7382, 7384, 7386, 7388, 7390, 7392, 7394,7396, 7398, 7400, 7402, 7404, 7406, 7408, 7410, 7412, 7414, 7416, 7418,7420, 7422, 7424, 7426, 7428, 7430, 7432, 7434, and 7436. In someembodiments, the polypeptide sequence of the engineeredglycosyltransferase comprises any of SEQ ID NOS: 7362, 7364, 7366, 7368,7370, 7372, 7374, 7376, 7378, 7380, 7382, 7384, 7386, 7388, 7390, 7392,7394, 7396, 7398, 7400, 7402, 7404, 7406, 7408, 7410, 7412, 7414, 7416,7418, 7420, 7422, 7424, 7426, 7428, 7430, 7432, 7434, and 7436.

The present invention also provides engineered glycosyltransferases,wherein the polypeptide sequences of the engineered glycosyltransferasescomprise at least one mutation or mutation set at one or more positionsselected from 2/3/433/435/442, 2/81, 9/37/113/396, 9/87, 9/144/331,37/113/144/396, 37/233, 53/144/233/269/331/428, 53/233/269/390/395/396,53/234, 61/120/156/163/165/197/303/308, 61/120/159/300/308/407,61/120/300/303/407, 61/156/159/163/165/243/248/253/300/303/308,61/156/163/243/248/300/303/308/407, 61/156/163/300/365/411,61/156/197/253, 61/163/300/303/308/405/407/411, 61/165/248/253/407/411,61/214, 61/214/300/308, 61/243/300/308/407/411, 61/300/303/308,61/300/303/405, 61/365/405, 69, 69/81, 69/134, 69/263,69/263/434/438/439, 69/439, 81, 81/134, 81/222, 81/222/263/322/435/442,81/433/435/438/442, 85/156/159/243/248/253/308/405/407/411, 87/144,87/144/396, 113/233/234, 120/156/159/169/197/214/303/308/365/405/407,120/156/159/248/300/308, 120/156/248/303/308/411, 120/159,120/159/165/197, 120/159/197/308/407/411, 120/159/197/365/411,120/197/253/300/308, 120/253/300/303/308/407, 120/308/407/411,134/222/263, 144/234/269, 156/165/248/300/303/308, 156/197/248/300/411,156/214/308/411, 156/248/253/308,159/163/165/197/214/243/300/303/308/407, 163/197/253/300/303/308/365,171/263, 183/233/234/331/428, 197/253/308/407, 197/300/303/308/365,197/300/308/411, 222, 222/263/435/442, 233/396/399, 263, 269,269/428/437, 300/303/308, 300/308/405/411, 308, and 322, wherein thepositions are numbered with reference to SEQ ID NO: 7388. In someembodiments, the polypeptide sequence of the engineeredglycosyltransferase comprises at least one mutation or mutation set atone or more positions selected from 2K/3M/433A/435E/442F, 2K/81T,9G/37S/113D/396T, 9G/87K, 9G/144Q/331R, 37S/113D/144Q/396T, 37S/2335,53E/144Q/233Q/269R/331R/428I, 53E/233Q/269R/390I/395I/396T, 53E/234N,61Q/120V/156S/163A/165L/197K/303V/308L, 61Q/120V/159K/300A/308L/407I,61Q/120V/300A/303V/407I,61Q/156S/159K/163A/165L/243I/248W/253T/300A/303V/308L,61Q/156S/163A/243I/248W/300A/303V/308L/407I,61Q/156S/163A/300A/365I/411T, 61Q/156S/197K/253E,61Q/163A/300A/303V/308L/405P/407I/411T, 61Q/165L/248W/253T/407I/411T,61Q/214R, 61Q/214R/300A/308L, 61Q/243I/300A/308L/407I/411T,61Q/300A/303V/308L, 61Q/300A/303V/405P, 61Q/365I/405P, 69A, 69A/81T,69A/134E, 69A/263T, 69A/263T/434E/438S/439H, 69A/439H, 81T, 81T/134E,81T/222A, 81T/222A/263T/322S/435E/442F, 81T/433A/435I/438R/442F,85V/156S/159K/243I/248W/253T/308L/405P/407I/411T, 87K/144Q,87K/144Q/396T, 113D/233Q/234N,120V/156/159K/169D/197K/214R/303V/308L/365I/405P/407I,120V/156S/159K/248W/300A/308L, 120V/156S/248W/303V/308L/411T, 120V/159K,120V/159K/165L/197K, 120V/159K/197K/308L/407I/411T,120V/159K/197K/365I/411T, 120V/197K/253T/300A/308L,120V/253T/300A/303V/308L/407I, 120V/308L/407I/411T, 134E/222A/263T,144Q/234N/269R, 1565/165L/248W/300A/303V/308L, 156S/197K/248W/300A/411T,1565/214R/308L/411T, 156S/248W/253T/308L,159K/163A/165L/197K/214R/243I/300A/303V/308L/407I,163A/197K/253E/300A/303V/308L/365I, 171P/263T, 183L/233Q/234N/331R/428I,197K/253T/308L/407I, 197K/300A/303V/308L/365I, 197K/300A/308L/411T,222A, 222A/263T/435I/442F, 233Q/396T/399Q, 263T, 269R, 269R/428I/437L,300A/303V/308L, 300A/308L/405P/411T, 308L, and 322S, wherein thepositions are numbered with reference to SEQ ID NO: 7388. In someembodiments, the polypeptide sequence of the engineeredglycosyltransferase comprises at least one mutation or mutation set atone or more positions selected from P2K/N3M/L433A/R435E/S442F, P2K/H81T,V9G/R37S/S113D/Y396T, V9G/M87K, V9G/M144Q/K331R, R37S/S113D/M144Q/Y396T,R37S/W233S, K53E/M144Q/W233Q/Q269R/K331R/K428I,K53E/W233Q/Q269R/V390I/V395I/Y396T, K53E/R234N,S61Q/L120V/C156S/L163A/Y165L/Q197K/Q303V/R308L,S61Q/L120V/Q159K/D300A/R308L/V407I, S61Q/L120V/D300A/Q303V/V407I,S61Q/C156S/Q159K/L163A/Y165L/L243I/H248W/S253T/D300A/Q303V/R308L,S61Q/C156S/L163A/L243I/H248W/D300A/Q303V/R308L/V407I,S61Q/C156S/L163A/D300A/V365I/R411T, S61Q/C156S/Q197K/S253E,S61Q/L163A/D300A/Q303V/R308L/E405P/V407I/R411T,S61Q/Y165L/H248W/S253T/V407I/R411T, S61Q/S214R, S61Q/S214R/D300A/R308L,S61Q/L243I/D300A/R308L/V407I/R411T, S61Q/D300A/Q303V/R308L,S61Q/D300A/Q303V/E405P, S61Q/V365I/E405P, D69A, D69A/H81T, D69A/D134E,D69A/V263T, D69A/V263T/D434E/A438S/P439H, D69A/P439H, H81T, H81T/D134E,H81T/K222A, H81T/K222A/V263T/L322S/R435E/S442F,H81T/L433A/R435I/A438R/S442F,A85V/C156S/Q159K/L243I/H248W/S253T/R308L/E405P/V407I/R411T, M87K/M144Q,M87K/M144Q/Y396T, S113D/W233Q/R234N,L120V/C156S/Q159K/E169D/Q197K/S214R/Q303V/R308L/V365I/E405P/V407I,L120V/C156S/Q159K/H248W/D300A/R308L,L120V/C156S/H248W/Q303V/R308L/R411T, L120V/Q159K,L120V/Q59K/Y165L/Q197K, L120V/Q59K/Q197K/R308L/V407I/R411T,L120V/Q159K/Q197K/V365I/R411T, L120V/Q197K/S253T/D300A/R308L,L120V/S253T/D300A/Q303V/R308L/V407I, L120V/R308L/V407I/R411T,D134E/K222A/V263T, M144Q/R234N/Q269R,C156S/Y165L/H248W/D300A/Q303V/R308L, C156S/Q197K/H248W/D300A/R411T,C156S/S214R/R308L/R411T, C156S/H248W/S253T/R308L,Q159K/L163A/Y165L/Q197K/S214R/L243I/D300A/Q303V/R308L/V407I,L163A/Q197K/S253E/D300A/Q303V/R308L/V365I, K171P/V263T,M183L/W233Q/R234N/K331R/K428I, Q197K/S253T/R308L/V407I,Q197K/D300A/Q303V/R308L/V365I, Q197K/D300A/R308L/R411T, K222A,K222A/V263T/R435I/S442F, W233Q/Y396T/N399Q, V263T, Q269R,Q269R/K428I/1437L, D300A/Q303V/R308L, D300A/R308L/E405P/R411T, R308L,and L322S, wherein the positions are numbered with reference to SEQ IDNO: 7388. In some embodiments, the polypeptide sequence of theengineered glycosyltransferase comprises at least one mutation ormutation set at one or more positions selected from 41, 56, 61, 72, 76,87, 88, 107, 139, 156, 338, and 407, wherein the positions are numberedwith reference to SEQ ID NO: 7388. In some embodiments, the polypeptidesequence of the engineered glycosyltransferase comprises at least onemutation or mutation set at one or more positions selected from 41E,56D, 61D, 61E, 72P, 76S, 87E, 88L, 88M, 107L, 107V, 139N, 156S, 338V,and 407T, wherein the positions are numbered with reference to SEQ IDNO: 7388. In some embodiments, the polypeptide sequence of theengineered glycosyltransferase comprises at least one mutation ormutation set at one or more positions selected from A41E, L56D, S61D,S61E, T72P, R76S, M87E, R88L, R88M, A107L, A107V, K139N, C156S, C338V,and V407T, wherein the positions are numbered with reference to SEQ IDNO: 7388. In some embodiments, the polypeptide sequence of theengineered glycosyltransferase comprises a sequence at least 90%identical to any of SEQ ID NOS: 7946, 7948, 7950, 7952, 7954, 7956,7958, 7960, 7962, 7964, 7966, 7968, 7970, 7972, 7974, 7976, 7978, 7980,7982, 7984, 7986, 7988, 7990, 7992, 7994, 7996, 7998, 8000, 8002, 8004,8006, 8008, 8010, 8012, 8014, 8016, 8018, 8020, 8022, 8024, 8026, 8028,8030, 8032, 8034, 8036, 8038, 8040, 8042, 8044, 8046, 8048, 8050, 8052,8054, 8056, 8058, 8060, 8062, 8064, 8066, 8068, 8070, 8072, 8074, 8076,8078, 8080, 8082, 8084, 8086, 8088, 8090, 8340, 8342, 8344, 8346, 8348,8350, 8352, 8354, 8356, 8358, 8360, 8362, 8364, 8366, and 8368. In someembodiments, the polypeptide sequence of the engineeredglycosyltransferase comprises a sequence at least 95% identical to anyof SEQ ID NOS: 7946, 7948, 7950, 7952, 7954, 7956, 7958, 7960, 7962,7964, 7966, 7968, 7970, 7972, 7974, 7976, 7978, 7980, 7982, 7984, 7986,7988, 7990, 7992, 7994, 7996, 7998, 8000, 8002, 8004, 8006, 8008, 8010,8012, 8014, 8016, 8018, 8020, 8022, 8024, 8026, 8028, 8030, 8032, 8034,8036, 8038, 8040, 8042, 8044, 8046, 8048, 8050, 8052, 8054, 8056, 8058,8060, 8062, 8064, 8066, 8068, 8070, 8072, 8074, 8076, 8078, 8080, 8082,8084, 8086, 8088, 8090, 8340, 8342, 8344, 8346, 8348, 8350, 8352, 8354,8356, 8358, 8360, 8362, 8364, 8366, and 8368. In some embodiments, thepolypeptide sequence of the engineered glycosyltransferase comprises anyof SEQ ID NOS: 7946, 7948, 7950, 7952, 7954, 7956, 7958, 7960, 7962,7964, 7966, 7968, 7970, 7972, 7974, 7976, 7978, 7980, 7982, 7984, 7986,7988, 7990, 7992, 7994, 7996, 7998, 8000, 8002, 8004, 8006, 8008, 8010,8012, 8014, 8016, 8018, 8020, 8022, 8024, 8026, 8028, 8030, 8032, 8034,8036, 8038, 8040, 8042, 8044, 8046, 8048, 8050, 8052, 8054, 8056, 8058,8060, 8062, 8064, 8066, 8068, 8070, 8072, 8074, 8076, 8078, 8080, 8082,8084, 8086, 8088, 8090, 8340, 8342, 8344, 8346, 8348, 8350, 8352, 8354,8356, 8358, 8360, 8362, 8364, 8366, and 8368.

The present invention also provides engineered glycosyltransferases,wherein the polypeptide sequences of the engineered glycosyltransferasescomprise at least one mutation or mutation set at one or more positionsselected from 37/72/76/81, 37/72/76/107/156/331, 37/72/195/331,53/81/195/197, 69/72/76/107, 72/76/107/156, 72/76/107/195/197, 72/269,81/107/195, 87, 87/91, 87/91/94, 87/91/94/120, 87/91/94/233,87/91/94/233/259, 87/91/94/263/389, 87/91/120/233,87/91/120/233/411/431/435/437, 87/91/144/259, 87/91/163, 87/91/163/233,87/91/163/233/263, 87/91/163/389, 87/91/233, 87/91/233/389,87/91/259/263/389/428/431/435/437, 87/91/263/389, 87/91/322, 87/91/389,87/94, 87/94/144/263/428/435, 87/94/263, 87/163/233, 87/233, 87/233/259,87/233/322/389/411, 87/233/389, 87/259, 87/263, 87/428/431/435, 87/435,91, 91/94, 91/94/120/233, 91/94/120/233/389/431/438, 91/94/233/259,91/120, 91/120/233, 91/233, 91/233/259/389, 91/233/389, 94/233/411,144/389, 163/233, 195/197, 197, 233, 233/259/263, 233/259/389, 233/438,259, 263, 263/389, 322, 389, and 428/431/435, wherein the positions arenumbered with reference to SEQ ID NO: 8088. In some embodiments, thepolypeptide sequence of the engineered glycosyltransferase comprises atleast one mutation or mutation set at one or more positions selectedfrom 37S/72P/76S/81T, 37S/72P/76S/107L/156S/331R, 37S/72P/195Q/331R,53E/81T/195Q/197K, 69A/72P/76S/107L, 72P/76S/107L/156S,72P/76S/107L/195Q/197K, 72P/269R, 81T/107L/195Q, 87A, 87A/91L,87A/91L/94C/233Q, 87A/91L/94C/233Q/259T, 87A/91L/120L/233Q,87A/91L/120L/233Q/411T/431M/435E/437L, 87A/91L/163A, 87A/91L/233Q,87A/91L/233Q/389L, 87A/91L/259T/263T/389L/428I/431M/435I/437L,87A/91L/322S, 87A/94C, 87A/94C/263T, 87A/233Q, 87A/233Q/259T,87A/233Q/322S/389L/411T, 87A/233Q/389L, 87A/259T, 87A/263T,87A/428I/431M/435E, 87A/435E, 87K, 87K/91L, 87K/91L/94C,87K/91L/94C/120L, 87K/91L/94C/233Q, 87K/91L/94C/263T/389L,87K/91L/120L/233Q, 87K/91L/144Q/259T, 87K/91L/163A/233Q,87K/91L/163A/233Q/263T, 87K/91L/163A/389L, 87K/91L/233Q,87K/91L/263T/389L, 87K/91L/389L, 87K/94C/144Q/263T/428I/435E,87K/163A/233Q, 91L, 91L/94C, 91L/94C/120L/233Q,91L/94C/120L/233Q/389L/431M/438S, 91L/94C/233Q/259T, 91L/120L,91L/120L/233Q, 91L/233Q, 91L/233Q/259T/389L, 91L/233Q/389L,94C/233Q/411T, 144Q/389L, 163A/233Q, 195Q/197K, 197K, 233Q,233Q/259T/263T, 233Q/259T/389L, 233Q/438S, 259T, 263T, 263T/389L, 3225,389L, and 428I/431M/435E, wherein the positions are numbered withreference to SEQ ID NO: 8088. In some embodiments, the polypeptidesequence of the engineered glycosyltransferase comprises at least onemutation or mutation set at one or more positions selected fromR37S/T72P/R76S/H81T, R37S/T72P/R76S/A107L/C156S/K331R,R37S/T72P/H195Q/K331R, K53E/H81T/H195Q/Q197K, D69A/T72P/R76S/A107L,T72P/R76S/A107L/C156S, T72P/R76S/A107L/H195Q/Q197K, T72P/Q269R,H81T/A107L/H195Q, M87A, M87A/Q91L, M87A/Q91L/K94C/W233Q,M87A/Q91L/K94C/W233Q/E259T, M87A/Q91L/V120L/W233Q,M87A/Q91L/V120L/W233Q/R411T/D431M/R435E/I437L, M87A/Q91L/L163A,M87A/Q91L/W233Q, M87A/Q91L/W233Q/E389L,M87A/Q91L/E259T/V263T/E389L/K428I/D431M/R435I/I437L, M87A/Q91L/L322S,M87A/K94C, M87A/K94C/V263T, M87A/W233Q, M87A/W233Q/E259T,M87A/W233Q/L322S/E389L/R411T, M87A/W233Q/E389L, M87A/E259T, M87A/V263T,M87A/K428I/D431M/R435E, M87A/R435E, M87K, M87K/Q91L, M87K/Q91L/K94C,M87K/Q91L/K94C/V120L, M87K/Q91L/K94C/W233Q, M87K/Q91L/K94C/V263T/E389L,M87K/Q91L/V120L/W233Q, M87K/Q91L/M144Q/E259T, M87K/Q91L/L163A/W233Q,M87K/Q91L/L163A/W233Q/V263T, M87K/Q91L/L163A/E389L, M87K/Q91L/W233Q,M87K/Q91L/V263T/E389L, M87K/Q91L/E389L,M87K/K94C/M144Q/V263T/K428I/R435E, M87K/L163A/W233Q, Q91L, Q91L/K94C,Q91L/K94C/V120L/W233Q, Q91L/K94C/V120L/W233Q/E389L/D431M/A438S,Q91L/K94C/W233Q/E259T, Q91L/V120L, Q91L/V120L/W233Q, Q91L/W233Q,Q91L/W233Q/E259T/E389L, Q91L/W233Q/E389L, K94C/W233Q/R411T, M144Q/E389L,L163A/W233Q, H195Q/Q197K, Q197K, W233Q, W233Q/E259T/V263T,W233Q/E259T/E389L, W233Q/A438S, E259T, V263T, V263T/E389L, L322S, E389L,and K428I/D431M/R435E, wherein the positions are numbered with referenceto SEQ ID NO: 8088. In some embodiments, the polypeptide sequence of theengineered glycosyltransferase comprises at least one mutation ormutation set at one or more positions selected from 55, 111, 252, 255,324, 328, 413, and 451, wherein the positions are numbered withreference to SEQ ID NO: 8088. In some embodiments, the polypeptidesequence of the engineered glycosyltransferase comprises at least onemutation or mutation set at one or more positions selected from 55G,111T, 252P, 255T, 324D, 324G, 328T, 413L, and 451Q, wherein thepositions are numbered with reference to SEQ ID NO: 8088. In someembodiments, the polypeptide sequence of the engineeredglycosyltransferase comprises at least one mutation or mutation set atone or more positions selected from S55G, S111T, S252P, S255T, P324D,P324G, L328T, V413L, and V451Q, wherein the positions are numbered withreference to SEQ ID NO: 8088. In some embodiments, the polypeptidesequence of the engineered glycosyltransferase comprises a sequence atleast 90% identical to any of SEQ ID NOS: 8482, 8484, 8486, 8488, 8490,8492, 8494, 8496, 8498, 8500, 8502, 8504, 8506, 8508, 8510, 8512, 8514,8516, 8518, 8520, 8522, 8524, 8526, 8528, 8530, 8532, 8534, 8536, 8538,8540, 8542, 8544, 8546, 8548, 8550, 8552, 8554, 8556, 8558, 8560, 8562,8564, 8566, 8568, 8570, 8572, 8574, 8576, 8578, 8580, 8582, 8584, 8586,8588, 8590, 8592, 8594, 8596, 8598, 8600, 8602, 8604, 8606, 8608, 8610,8612, 8614, 8616, 8618, 8620, 8622, 9224, 9226, 9228, 9230, 9232, 9234,9236, 9238, and 9240. In some embodiments, the polypeptide sequence ofthe engineered glycosyltransferase comprises a sequence at least 95%identical to any of SEQ ID NOS: 8482, 8484, 8486, 8488, 8490, 8492,8494, 8496, 8498, 8500, 8502, 8504, 8506, 8508, 8510, 8512, 8514, 8516,8518, 8520, 8522, 8524, 8526, 8528, 8530, 8532, 8534, 8536, 8538, 8540,8542, 8544, 8546, 8548, 8550, 8552, 8554, 8556, 8558, 8560, 8562, 8564,8566, 8568, 8570, 8572, 8574, 8576, 8578, 8580, 8582, 8584, 8586, 8588,8590, 8592, 8594, 8596, 8598, 8600, 8602, 8604, 8606, 8608, 8610, 8612,8614, 8616, 8618, 8620, 8622, 9224, 9226, 9228, 9230, 9232, 9234, 9236,9238, and 9240. In some embodiments, the polypeptide sequence of theengineered glycosyltransferase comprises any of SEQ ID NOS: 8482, 8484,8486, 8488, 8490, 8492, 8494, 8496, 8498, 8500, 8502, 8504, 8506, 8508,8510, 8512, 8514, 8516, 8518, 8520, 8522, 8524, 8526, 8528, 8530, 8532,8534, 8536, 8538, 8540, 8542, 8544, 8546, 8548, 8550, 8552, 8554, 8556,8558, 8560, 8562, 8564, 8566, 8568, 8570, 8572, 8574, 8576, 8578, 8580,8582, 8584, 8586, 8588, 8590, 8592, 8594, 8596, 8598, 8600, 8602, 8604,8606, 8608, 8610, 8612, 8614, 8616, 8618, 8620, 8622, 9224, 9226, 9228,9230, 9232, 9234, 9236, 9238, and 9240.

In some embodiments, the polypeptide sequence of the engineeredglycosyltransferase comprises a sequence at least 90% identical to anyof SEQ ID NOS:76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102,104, 106, and/or 108. In some embodiments, the polypeptide sequence ofthe engineered glycosyltransferase comprises a sequence at least 95%identical to any of SEQ ID NOS: 76, 78, 80, 82, 84, 86, 88, 90, 92, 94,96, 98, 100, 102, 104, 106, and/or 108. In some embodiments, thepolypeptide sequence of the engineered glycosyltransferase comprises SEQID NOS: 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104,106, and/or 108.

The present invention also provides engineered glycosyltransferases,wherein the polypeptide sequences of the engineered glycosyltransferasescomprise at least one mutation or mutation set at one or more positionsselected from 69/173/175/243/246/354/365/383/399, 69/173/243/383/399,56/191/354/383/399, 70/225/246/409/413, 70/115/225/409, 70/225/413,70/225/247, 74/310/396/424, 74/396, and 173/175/191/365/383/399, whereinthe positions are numbered with reference to SEQ ID NO:758. In someembodiments, the polypeptide sequence of the engineeredglycosyltransferase comprises at least one mutation or mutation set atone or more positions selected from69H/173N/1755/243A/246K/354I/365I/383V/399A, 69H/173N/243A/383V/399A,56T/191D/354I/383V/399A, 70L/225G/246P/409K/413V, 70L/115S/225G/409K,70L/225G/413V, 70L/225G/247G, 74T/310D/396E/4245, 74T/396E, and173H/175S/191D/365I/383V/399A, wherein the positions are numbered withreference to SEQ ID NO:758. In some embodiments, the polypeptidesequence of the engineered glycosyltransferase comprises at least onemutation or mutation set at one or more positions selected fromR69H/Y173N/P175S/V243A/M246K/L354I/M365I/M383V/T399A,R69H/Y173N/V243A/M383V/T399A, I56T/N191D/L354I/M383V/T399A,F70L/N225G/M246P/E409K/I413V, F70L/Q115S/N225G/E409K, F70L/N225G/I413V,F70L/N225G/E247G, H74T/K310D/G396E/N424S, H74T/G396E, andY173H/P175S/N191D/M365I/M383V/T399A, wherein the positions are numberedwith reference to SEQ ID NO:758. In some embodiments, the polypeptidesequence of the engineered glycosyltransferase comprises a sequence atleast 90% identical to any of SEQ ID NOS: 770, 772, 774, 776, 778, 780,782, 784, 786, and/or 788. In some embodiments, the polypeptide sequenceof the engineered glycosyltransferase comprises a sequence at least 95%identical to any of SEQ ID NOS: 770, 772, 774, 776, 778, 780, 782, 784,786, and/or 788. In some additional embodiments, the polypeptidesequence of the engineered glycosyltransferase comprises any of SEQ IDNOS: 770, 772, 774, 776, 778, 780, 782, 784, 786, and/or 788.

The present invention also provides engineered glycosyltransferases,wherein the polypeptide sequences of the engineered glycosyltransferasescomprise at least one mutation or mutation set at one or more positionsselected from 24/28, 24/28/262, 24/28/262/264, 24/28/262/264/423,24/264/294, 28, 28/262/264, 28/423, 69/173/175/243/246/354/365/383/399,69/173/243/383/399, 56/191/354/383/399, 70/115/225/409,70/225/246/409/413, 70/225/247, 70/225/413, 74/310/396/424, 74/396, 159,173/175/191/365/383/399, 199, 262/264, and 264/29, wherein the positionsare numbered with reference to SEQ ID NO:770. In some embodiments, thepolypeptide sequence of the engineered glycosyltransferase comprises atleast one mutation or mutation set at one or more positions selectedfrom 24E/28N, 24E/28N/262Y, 24E/28N/262Y/264S, 24E/28N/262Y/264S/423E,24E/264S/294V, 28N, 28N/262Y/264S, 28N/423E,69H/173N/175/243A/246K/354I/365I/383V/399A, 69H/173N/243A/383V/399A,56T/191D/354I/383V/399A, 70L/1155/225G/409K, 70L/225G/246P/409K/413V,70L/225G/247G, 70L/225G/413V, 74T/310D/396E/4245, 74T/396E, 159R,173H/1755/191D/365I/383V/399A, 199H, 262Y/264S, and 2645/291V, whereinthe positions are numbered with reference to SEQ ID NO:770. In someembodiments, the polypeptide sequence of the engineeredglycosyltransferase comprises at least one mutation or mutation set atone or more positions selected from Y24E/S28N, Y24E/S28N/F262Y,Y24E/S28N/F262Y/C264S, Y24E/S28N/F262Y/C264S/K423E, Y24E/C264S/A294V,S28N, S28N/F262Y/C264S, S28N/K423E,R69H/Y173N/P175S/V243A/M246K/L354I/M365I/M383V/T399A,R69H/Y173N/V243A/M383V/T399A, I56T/N191D/L354I/M383V/T399A,F70L/Q115S/N225G/E409K, F70L/N225G/M246P/E409K/I413V, F70L/N225G/E247G,F70L/N225G/I413V, H74T/K310D/G396E/N424S, H74T/G396E, F156R,Y173H/P175S/N191D/M365I/M383V/T399A, G199H, F262Y/C264S, andC264S/I291V, wherein the positions are numbered with reference to SEQ IDNO:770. In some embodiments, the polypeptide sequence of the engineeredglycosyltransferase comprises a sequence at least 90% identical to anyof SEQ ID NOS: 790, 792, 794, 796, 798, 800, 802, 804, 806, 808, 1292,and/or 1294. In some embodiments, the polypeptide sequence of theengineered glycosyltransferase comprises a sequence at least 95%identical to any of SEQ ID NOS: 790, 792, 794, 796, 798, 800, 802, 804,806, 808, 1292, and/or 1294. In some additional embodiments, thepolypeptide sequence of the engineered glycosyltransferase comprises anyof SEQ ID NOS: 790, 792, 794, 796, 798, 800, 802, 804, 806, 808, 1292,and/or 1294.

The present invention also provides engineered glycosyltransferases,wherein the polypeptide sequences of the engineered glycosyltransferasescomprise at least one mutation or mutation set at one or more positionsselected from 24, 28, 32, 264, 269, 325, 341, 351, and 366, wherein thepositions are numbered with reference to SEQ ID NO:770. In someembodiments, the polypeptide sequence of the engineeredglycosyltransferase comprises at least one mutation or mutation set atone or more positions selected from 24L/V, 28G/K/L, 32C/R/S, 264A/G,269S/W, 325G/H, 341V, 351L, and 366L/Q/T, wherein the positions arenumbered with reference to SEQ ID NO:770. In some embodiments, thepolypeptide sequence of the engineered glycosyltransferase comprises atleast one mutation or mutation set at one or more positions selectedfrom Y24L/V, S28G/K/L, N32C/R/S, C264A/G, Y269S/W, K325G/H, F341V,M351L, and H366L/Q/T, wherein the positions are numbered with referenceto SEQ ID NO:770. In some embodiments, the polypeptide sequence of theengineered glycosyltransferase comprises a sequence at least 90%identical to any of SEQ ID NOS: 810, 812, 814, 816, 818, 820, 822, 824,826, 828, 830, 832, 834, 836, 838, 840, 842, 844, and/or 846.

In some embodiments, the polypeptide sequence of the engineeredglycosyltransferase comprises a sequence at least 95% identical to anyof SEQ ID NOS: 810, 812, 814, 816, 818, 820, 822, 824, 826, 828, 830,832, 834, 836, 838, 840, 842, 844, and/or 846. In some embodiments, thepolypeptide sequence of the engineered glycosyltransferase comprises anyof SEQ ID NOS: 810, 812, 814, 816, 818, 820, 822, 824, 826, 828, 830,832, 834, 836, 838, 840, 842, 844, and/or 846.

The present invention also provides engineered glycosyltransferases,wherein the polypeptide sequences of the engineered glycosyltransferasescomprise at least one mutation or mutation set at one or more positionsselected from 56/69/70/175/191/246, 56/69/70/175/191/246/354,56/69/70/175/354, 56/69/70/191/246/354/365, 56/69/70/246/354,56/69/175/191/246/354, 56/69/175/246/354, 56/69/175/246/354/365,56/69/246/354, 56/69/246/365, 56/70/175/191/246, 56/70/175/191/246/354,56/70/175/191/354/365, 56/70/175/246/354, 56/70/175/246/365,56/70/191/246/354/365, 56/70/191/354, 56/175/246, 56/175/354,56/175/354/365, 56/191/246/354, 56/246, 56/246/354/365, 56/354, 69,69/70/175/191/246/354/365, 69/70/191/246/354/365, 69/70/246,69/70/354/365, 69/175/191/246/354, 69/175/354, 69/246/354/365, 69/354,70, 70/175/191/246/354/365, 70/175/191/354/365, 70/191/246/354/365,70/191/246/365, 126, 126/220, 126/403, 175, 175/191,175/191/246/354/365, 175/191/354, 175/191/354/365, 175/246/354,175/246/354/365, 175/354, 191/246/354, 246/354, 354, and 354/365,wherein the positions are numbered with reference to SEQ ID NO:770. Insome embodiments, the polypeptide sequence of the engineeredglycosyltransferase comprises at least one mutation or mutation set atone or more positions selected from 56T/69Q/70L/175S/191D/246K,56T/69Q/70L/175S/191D/246K/354I, 56T/69Q/70L/175S/354I,56T/69Q/70L/191D/246K/354I/365I, 56T/69Q/70L/246K/354I,56T/69Q/175S/191D/246K/354I, 56T/69Q/175S/246K/354I,56T/69Q/175S/246K/354I/365I, 56T/69Q/246K/354I, 56T/69Q/246K/M65I,56T/70L/175S/191D/246K, 56T/70L/175S/191D/246K/354I,56T/70L/175S/191D/354I/365I, 56T/70L/175S/246K/354I,56T/70L/175S/246K/365I, 56T/70L/191D/246K/354I/365I, 56T/70L/191D/354I,56T/175S/246K, 56T/175S/354I, 56T/175S/354I/365I, 56T/191D/246K/354I,56T/246K, 56T/246K/354I/365I, 56T/354I, 69Q,69Q/70L/175S/191D/246K/354I/365I, 69Q/70L/191D/246K/354I/365I,69Q/70L/246K, 69Q/70L/354I/365I, 69Q/175S/191D/246K/354I, 69Q/175S/354I,69Q/246K/354I/365I, 69Q/354I, 70L, 70L/175S/191D/246K/354I/365I,70L/175S/191D/354I/365I, 70L/191D/246K/354I/365I, 70L/191D/246K/365I,126F, 126F/220L, 126F/403R, 175S, 175/191D, 175/191D/246K/354I/365I,175S/191D/354I, 175S/191D/354I/365I, 175S/246K/354I,175S/246K/354I/365I, 175S/354I, 191D/246K/354I, 246K/354I, 354I, and354I/365I, wherein the positions are numbered with reference to SEQ IDNO:770. In some embodiments, the polypeptide sequence of the engineeredglycosyltransferase comprises at least one mutation or mutation set atone or more positions selected from I56T/H69Q/F70L/P175S/N191D/M246K,I56T/H69Q/F70L/P175S/N191D/M246K/L354I, I56T/H69Q/F70L/P175S/L354I,I56T/H69Q/F70L/N191D/M246K/L354I/M365I, I56T/H69Q/F70L/M246K/L354I,I56T/H69Q/P175S/N191D/M246K/L354I, I56T/H69Q/P175S/M246K/L354I,I56T/H69Q/P175S/M246K/L354I/M365I, I56T/H69Q/M246K/L354I,156T/H69Q/M246K/M365I, I56T/F70L/P175S/N191D/M246K,I56T/F70L/P175S/N191D/M246K/L354I, I56T/F70L/P175S/N191D/L354I/M365I,I56T/F70L/P175S/M246K/L354I, 156T/F70L/P175S/M246K/M365I,I56T/F70L/N191D/M246K/L354I/M365I, I56T/F70L/N191D/L354I,I56T/P175S/M246K, I56T/P175S/L354I, 156T/P175S/L354I/M365I,156T/N191D/M246K/L354I, I56T/M246K, 156T/M246K/L354I/M365I, I56T/L354I,H69Q, H69Q/F70L/P175S/N191D/M246K/L354I/M365I,H69Q/F70L/N191D/M246K/L354I/M365I, H69Q/F70L/M246K,H69Q/F70L/L354I/M365I, H69Q/P175S/N191D/M246K/L354I, H69Q/P175S/L354I,H69Q/M246K/L354I/M365I, H69Q/L354I, F70L,F70L/P175S/N191D/M246K/L354I/M365I, F70L/P175S/N191D/L354I/M365I,F70L/N191D/M246K/L354I/M365I, F70L/N191D/M246K/M365I, I126F,I126F/C220L, I126F/K403R, P175S, P175S/N191D,P175S/N191D/M246K/L354I/M365I, P175S/N191D/L354I,P175S/N191D/L354I/M365I, P175S/M246K/L354I, P175S/M246K/L354I/M365I,P175S/L354I, N191D/M246K/L354I, M246K/L354I, L354I, and L354I/M365I,wherein the positions are numbered with reference to SEQ ID NO:770. Insome additional embodiments, the polypeptide sequence of the engineeredglycosyltransferase comprises a sequence at least 90% identical to anyof SEQ ID NOS: 848, 850, 852, 854, 856, 858, 860, 862, 864, 866, 868,870, 872, 874, 876, 878, 880, 882, 884, 886, 888, 890, 892, 894, 896,898, 900, 902, 904, 906, 908, 910, 912, 914, 916, 918, 920, 922, 924,926, 928, 930, 932, 934, 936, 938, 940, 942, 944, 946, 948, 950, and/or952. In some embodiments, the polypeptide sequence of the engineeredglycosyltransferase comprises a sequence at least 95% identical to anyof SEQ ID NOS: 848, 850, 852, 854, 856, 858, 860, 862, 864, 866, 868,870, 872, 874, 876, 878, 880, 882, 884, 886, 888, 890, 892, 894, 896,898, 900, 902, 904, 906, 908, 910, 912, 914, 916, 918, 920, 922, 924,926, 928, 930, 932, 934, 936, 938, 940, 942, 944, 946, 948, 950, and/or952. In some embodiments, the polypeptide sequence of the engineeredglycosyltransferase comprises any of SEQ ID NOS: 848, 850, 852, 854,856, 858, 860, 862, 864, 866, 868, 870, 872, 874, 876, 878, 880, 882,884, 886, 888, 890, 892, 894, 896, 898, 900, 902, 904, 906, 908, 910,912, 914, 916, 918, 920, 922, 924, 926, 928, 930, 932, 934, 936, 938,940, 942, 944, 946, 948, 950, and/or 952.

The present invention also provides engineered glycosyltransferases,wherein the polypeptide sequences of the engineered glycosyltransferasescomprise at least one mutation or mutation set at one or more positionsselected from 24/32/264/269/330/402/403, 24/32/264/269/382/403/406,24/32/264/330, 24/32/269, 24/32/269/382/385/389/402/406, 24/32/269/403,24/32/330, 24/264/269/389/402/406, 70/126/175/191/246/325/354/366,70/126/175/325/330/351/354/366, 70/126/191/246/325/351/354/366/423,70/126/191/246/325/351/354/423, 70/126/191/246/325/351/366/423,70/126/191/246/354/366, 70/126/246/330/366, 70/126/246/354/366,126/211/220/253/316/342, 126/211/220/275/279/323, 126/211/260/423,126/220/260, 126/220/316, 56/220/260/423, 220/260, and 220/260/423,wherein the positions are numbered with reference to SEQ ID NO:792. Insome embodiments, the polypeptide sequence of the engineeredglycosyltransferase comprises at least one mutation or mutation set atone or more positions selected from 24L/32S/264A/269S/330Q/402V/403R,24L/32S/264A/269S/382G/403R/406M, 24L/32S/264A/330Q, 24L/32S/269S,24L/32S/269S/382G/385V/389E/402I/406M, 24L/32S/269S/403R, 24L/32S/330Q,24L/264A/269S/389E/402V/406M, 70L/126F/175S/191D/246K/325H/354I/366Q,70L/126F/175S/325H/330Q/351L/354I/366Q,70L/126F/191D/246K/325H/351L/354I/366Q/423K,70L/126F/191D/246K/325H/351L/354I/423K,70L/126F/191D/246K/325H/351L/366Q/423K, 70L/126F/191D/246K/354I/366Q,70L/126F/246K/330Q/366Q, 70L/126F/246K/354I/366Q,126F/211E/220L/253D/316V/342L, 126F/211E/220L/275Q/279L/323V,126F/211E/260V/423K, 126F/220L/260V, 126F/220L/316V, 56V/220L/260V/423K,220L/260V, and 220L/260V/423K, wherein the positions are numbered withreference to SEQ ID NO:792. In some embodiments, the polypeptidesequence of the engineered glycosyltransferase comprises at least onemutation or mutation set at one or more positions selected fromE24L/N32S/S264A/Y269S/P330Q/L402V/K403R,E24L/N32S/S264A/Y269S/A382G/K403R/I406M, E24L/N32S/S264A/P330Q,E24L/N32S/Y269S, E24L/N32S/Y269S/A382G/I385V/D389E/L402I/I406M,E24L/N32S/Y269S/K403R, E24L/N32S/P330Q,E24L/S264A/Y269S/D389E/L402V/I406M,F70L/I126F/P175S/N191D/M246K/K325H/L354I/H366Q,F70L/I126F/P175S/K325H/P330Q/M351L/L354I/H366Q,F70L/I126F/N191D/M246K/K325H/M351L/L354I/H366Q/E423K,F70L/I126F/N191D/M246K/K325H/M351L/L354I/E423K,F70L/I126F/N191D/M246K/K325H/M351L/H366Q/E423K,F70L/I126F/N191D/M246K/L354I/H366Q, F70L/I126F/M246K/P330Q/H366Q,F70L/I126F/M246K/L354I/H366Q, I126F/T211E/C220L/G253D/I316V/I342L,I126F/T211E/C220L/D275Q/V279L/L323V, I126F/T211E/T260V/E423K,I126F/C220L/T260V, I126F/C220L/I316V, I56V/C220L/T260V/E423K,C220L/T260V, and C220L/T260V/E423K, wherein the positions are numberedwith reference to SEQ ID NO:792. In some embodiments, the polypeptidesequence of the engineered glycosyltransferase comprises a sequence atleast 90% identical to any of SEQ ID NOS: 954, 956, 958, 960, 962, 964,966, 968, 970, 972, 974, 976, 978, 980, 982, 984, 986, 988, 990, 992,994, 996, 998, and/or 1000. In some embodiments, the polypeptidesequence of the engineered glycosyltransferase comprises a sequence atleast 95% identical to any of SEQ ID NOS: 954, 956, 958, 960, 962, 964,966, 968, 970, 972, 974, 976, 978, 980, 982, 984, 986, 988, 990, 992,994, 996, 998, and/or 1000. In some embodiments, the polypeptidesequence of the engineered glycosyltransferase comprises any of SEQ IDNOS: 954, 956, 958, 960, 962, 964, 966, 968, 970, 972, 974, 976, 978,980, 982, 984, 986, 988, 990, 992, 994, 996, 998, and/or 1000.

The present invention also provides engineered glycosyltransferases,wherein the polypeptide sequence of the engineered glycosyltransferasescomprise at least one mutation or mutation set at one or more positionsselected from 24/32/97/162/202, 24/32/126/198/201/367,24/32/126/198/202/220/226/260/2695, 24/32/146/226,24/32/198/201/220/226, 24/32/198/201/226, 24/97/260/367,32/97/198/202/226/260, 32/202/367, 32/226/367,56/175/197/211/330/382/385, 56/175/197/253/385/389,56/175/264/382/385/389/402/406, 56/197/264/279/330/382/389/402/403/406,56/211/253/316/323, 56/211/264/316/389, 56/211/279/323/330/402,56/264/385/389, 97/202/367, 162/220/226/367, 175/197/211/264/330,175/211/264/279/316/323, 175/211/279/323/330/402/403/406,175/211/323/382/402/403/406, 175/211/403/406,175/264/316/323/330/403/406, 175/264/316/389/402, 175/264/323/330,197/211/316/342/406, 197/211/316/382/389/402/403, 197/211/402,197/279/323, 198/201/367, 198/202/220/269/367, 201/202/367, 211/382/406,211/385/389, 211/402/403, and 389, wherein the positions are numberedwith reference to SEQ ID NO:954. In some embodiments, the polypeptidesequence of the engineered glycosyltransferase comprises at least onemutation or mutation set at one or more positions selected from24L/32S/97G/162R/202G, 24L/32S/126A/198P/201G/367W,24L/32S/126A/198P/202G/220L/226V/260V/269S, 24L/32S/146A/226V,24L/32S/198P/201G/220L/226V, 24L/32S/198P/201G/226V, 24L/97G/260V/367W,32S/97G/198P/202G/226V/260V, 32S/202G/367W, 32S/226V/367W,56V/175S/197P/211E/330Q/382G/385V, 56V/175S/197P/253D/385V/389E,56V/175S/264A/382G/385V/389E/402V/406M,56V/197P/264A/279L/330Q/382G/389E/402V/403R/406M,56V/211E/253D/316V/323V, 56V/211E/264A/316V/389E,56V/211E/279L/323V/330Q/402I, 56V/264A/385V/389E, 97G/202G/367W,162R/220L/226V/367W, 175S/197P/211E/264A/330Q,1755/211E/264A/279L/316V/323V, 1755/211E/279L/323V/330Q/402I/403R/406M,1755/211E/323V/382G/402I/403R/406M, 1755/211E/403R/406M,175S/264A/316V/323V/330Q/403R/406M, 175S/264A/316V/389E/402V,175S/264A/323V/330Q, 197P/211E/316V/342L/406M,197P/211E/316V/382G/389E/402I/403R, 197P/211E/402I, 197P/279L/323V,198P/201G/367W, 198P/202G/220L/269S/367W, 201G/202G/367W,211E/382G/406M, 211E/385V/389E, 211E/402I/403R, and D389E, wherein thepositions are numbered with reference to SEQ ID NO:954. In someembodiments, the polypeptide sequence of the engineeredglycosyltransferase comprises at least one mutation or mutation set atone or more positions selected from E24L/N32S/H97G/N162R/Q202G,E24L/N32S/F126A/E198P/M201G/N367W,E24L/N32S/F126A/E198P/Q202G/C220L/W226V/T260V/Y269S,E24L/N32S/L146A/W226V, E24L/N32S/E198P/M201G/C220L/W226V,E24L/N32S/E198P/M201G/W226V, E24L/H97G/T260V/N367W,N32S/H97G/E198P/Q202G/W226V/T260V, N32S/Q202G/N367W, N32S/W226V/N367W,I56V/P175S/A197P/T211E/P330Q/A382G/I385V,I56V/P175S/A197P/G253D/I385V/D389E,I56V/P175S/S264A/A382G/I385V/D389E/L402V/I406M,I56V/A197P/S264A/V279L/P330Q/A382G/D389E/L402V/K403R/I406M,I56V/T211E/G253D/I316V/L323V, 156V/T211E/S264A/I316V/D389E,I56V/T211E/V279L/L323V/P330Q/L402I, I56V/S264A/I385V/D389E,H97G/Q202G/N367W, N162R/C220L/W226V/N367W,P175S/A197P/T211E/S264A/P330Q, P175S/T211E/S264A/V279L/I316V/L323V,P175S/T211E/V279L/L323V/P330Q/L402I/K403R/I406M,P175S/T211E/L323V/A382G/L402I/K403R/I406M, P175S/T211E/K403R/I406M,P175S/S264A/I316V/L323V/P330Q/K403R/I406M,P175S/S264A/I316V/D389E/L402V, P175S/S264A/L323V/P330Q,A197P/T211E/I316V/I342L/I406M,A197P/T211E/I316V/A382G/D389E/L402I/K403R, A197P/T211E/L402I,A197P/V279L/L323V, E198P/M201G/N367W, E198P/Q202G/C220L/Y269S/N367W,M201G/Q202G/N367W, T211E/A382G/I406M, T211E/I385V/D389E,T211E/L402I/K403R, and D389E, wherein the positions are numbered withreference to SEQ ID NO: 954. In some embodiments, the polypeptidesequence of the engineered glycosyltransferase comprises a sequence atleast 90% identical to any of SEQ ID NOS:1002, 1004, 1006, 1008, 1010,1012, 1014, 1016, 1018, 1020, 1022, 1024, 1026, 1028, 1030, 1032, 1034,1036, 1038, 1040, 1042, 1044, 1046, 1048, 1050, 1052, 1054, 1056, 1058,1060, 1062, 1064, 1066, 1068, 1070, 1072, 1074, 1076, and/or 1078. Insome embodiments, the polypeptide sequence of the engineeredglycosyltransferase comprises a sequence at least 95% identical to anyof SEQ ID NOS: 1002, 1004, 1006, 1008, 1010, 1012, 1014, 1016, 1018,1020, 1022, 1024, 1026, 1028, 1030, 1032, 1034, 1036, 1038, 1040, 1042,1044, 1046, 1048, 1050, 1052, 1054, 1056, 1058, 1060, 1062, 1064, 1066,1068, 1070, 1072, 1074, 1076, and/or 1078. In some additionalembodiments, the polypeptide sequence of the engineeredglycosyltransferase comprises any of SEQ ID NOS: 1002, 1004, 1006, 1008,1010, 1012, 1014, 1016, 1018, 1020, 1022, 1024, 1026, 1028, 1030, 1032,1034, 1036, 1038, 1040, 1042, 1044, 1046, 1048, 1050, 1052, 1054, 1056,1058, 1060, 1062, 1064, 1066, 1068, 1070, 1072, 1074, 1076, and/or 1078.

The present invention also provides engineered glycosyltransferases,wherein the polypeptide sequence of the engineered glycosyltransferasescomprise at least one mutation or mutation set at one or more positionsselected from 24/32/175/211/260/330/403/406, 24/32/175/226/253/275/316,24/32/201/226/260/264/330/402/406,24/162/175/198/211/226/275/316/323/389, 24/162/175/201/275/316,24/162/198/201/211/226/323/351, 24/162/198/201/226/351,24/162/201/253/264/351/402/406, 24/175/198/211/226/253/316,24/175/201/275/316/351, 24/175/201/316, 24/175/211/220/260/275/330/389,24/175/211/253/316, 24/175/211/316/330, 24/175/226/323/351, 24/198/201,24/198/201/211, 24/198/201/211/220/260, 24/198/201/220/275/389/402/406,24/198/201/226/330/389, 24/198/201/351,24/201/211/253/323/351/366/389/402/403, 24/201/226/253/402/403/406,24/226/330/351/403, 175/198/201/211/226/260/264/323/402/406, and175/198/226/260/351/402/403/406, wherein the positions are numbered withreference to SEQ ID NO: 1054. In some embodiments, the polypeptidesequence of the engineered glycosyltransferase comprises at least onemutation or mutation set at one or more positions selected from24L/32S/175S/211E/260V/330Q/403R/406M, 24L/32S/175S/226V/253D/275Q/316V,24L/32S/201G/226V/260V/264A/330Q/402I/406M,24L/162R/175S/198P/211E/226V/275Q/316V/323V/389E,24L/162R/175S/201G/275Q/316V, 24L/162R/198P/201G/211E/226V/323V/351M,24L/162R/198P/201G/226V/351M, 24L/162R/201G/253D/264A/351M/402I/406M,24L/175S/198P/211E/226V/253D/316V, 24L/175S/201G/275Q/316V/351M,24L/175S/201G/316V, 24L/1755/211E/220L/260V/275Q/330Q/389E,24L/175S/211E/253D/316V, 24L/175S/211E/316V/330Q,24L/175S/226V/323V/351M, 24L/198P/201G, 24L/198P/201G/211E,24L/198P/201G/211E/220L/260V, 24L/198P/201G/220L/275Q/389E/402I/406M,24L/198P/201G/226V/330Q/389E, 24L/198P/201G/351M,24L/201G/211E/253D/323V/351M/366H/389E/402I/403R,24L/201G/226V/253D/402I/403R/406M, 24L/226V/330Q/351M/403R,175S/198P/201G/211E/226V/260V/264A/323V/402I/406M, and175S/198P/226V/260V/351M/402I/403R/406M, wherein the positions arenumbered with reference to SEQ ID NO: 1054. In some embodiments, thepolypeptide sequence of the engineered glycosyltransferase comprises atleast one mutation or mutation set at one or more positions selectedfrom E24L/N32S/P175S/T211E/T260V/P330Q/K403R/I406M,E24L/N32S/P175S/W226V/G253D/D275Q/I316V,E24L/N32S/M201G/W226V/T260V/S264A/P330Q/L402I/I406M,E24L/N162R/P175S/E198P/T211E/W226V/D275Q/I316V/L323V/D389E,E24L/N162R/P175S/M201G/D275Q/I316V,E24L/N162R/E198P/M201G/T211E/W226V/L323V/L351M,E24L/N162R/E198P/M201G/W226V/L351M,E24L/N162R/M201G/G253D/S264A/L351M/L402I/I406M,E24L/P175S/E198P/T211E/W226V/G253D/I316V,E24L/P175S/M201G/D275Q/I316V/L351M, E24L/P175S/M201G/I316V,E24L/P175S/T211E/C220L/T260V/D275Q/P330Q/D389E,E24L/P175S/T211E/G253D/I316V, E24L/P175S/T211E/I316V/P330Q,E24L/P175S/W226V/L323V/L351M, E24L/E198P/M201G, E24L/E198P/M201G/T211E,E24L/E198P/M201G/T211E/C220L/T260V,E24L/E198P/M201G/C220L/D275Q/D389E/L402I/I406M,E24L/E198P/M201G/W226V/P330Q/D389E, E24L/E198P/M201G/L351M,E24L/M201G/T211E/G253D/L323V/L351M/Q366H/D389E/L402I/K403R,E24L/M201G/W226V/G253D/L402I/K403R/I406M, E24L/W226V/P330Q/L351M/K403R,P175S/E198P/M201G/T211E/W226V/T260V/S264A/L323V/L402I/I406M, andP175S/E198P/W226V/T260V/L351M/L402I/K403R/I406M, wherein the positionsare numbered with reference to SEQ ID NO: 1054. In some embodiments, thepolypeptide sequence of the engineered glycosyltransferase comprises asequence at least 90% identical to any of SEQ ID NOS: 2596, 2598, 2600,2602, 2604, 2606, 2608, 2610, 2612, 2614, 2616, 2618, 2620, 2622, 2624,2626, 2628, 2630, 2632, 2634, 2636, 2638, 2640, 2642, 2644, and 2646. Insome embodiments, the polypeptide sequence of the engineeredglycosyltransferase comprises a sequence at least 95% identical to anyof SEQ ID NOS:: 2596, 2598, 2600, 2602, 2604, 2606, 2608, 2610, 2612,2614, 2616, 2618, 2620, 2622, 2624, 2626, 2628, 2630, 2632, 2634, 2636,2638, 2640, 2642, 2644, and 2646. In some embodiments, the polypeptidesequence of the engineered glycosyltransferase comprises any of SEQ IDNOS:: 2596, 2598, 2600, 2602, 2604, 2606, 2608, 2610, 2612, 2614, 2616,2618, 2620, 2622, 2624, 2626, 2628, 2630, 2632, 2634, 2636, 2638, 2640,2642, 2644, and 2646.

The present invention also provides engineered glycosyltransferases,wherein the polypeptide sequence of the engineered glycosyltransferasescomprise at least one mutation or mutation set at one or more positionsselected from 21/127/129/161, 21/127/129/161/162,21/127/129/162/199/200, 127/129/161/162/199, 127/129/161/199/200,127/129/162, 156, 156/161, 156/161/162, 156/162/199, and 156/199/200,wherein the positions are numbered with reference to SEQ ID NO: 1002. Insome embodiments, the polypeptide sequence of the engineeredglycosyltransferase comprises at least one mutation or mutation set atone or more positions selected from 21Y/127H/129A/161S,21Y/127H/129A/161S/162G, 21Y/127H/129A/162T/199H/200A, 127H/129A/162T,127Q/129A/161S/162G/199H, 127Q/129A/161S/199H/200A, 156R, 156R/161S,156R/161S/162G, 156R/161S/162T, 156R/162G/199H, and 156R/199H/200A,wherein the positions are numbered with reference to SEQ ID NO: 1002. Insome embodiments, the polypeptide sequence of the engineeredglycosyltransferase comprises at least one mutation or mutation set atone or more positions selected from W21Y/L127H/P129A/K161S,W21Y/L127H/P129A/K161S/N162G, W21Y/L127H/P129A/N162T/G199H/N200A,L127H/P129A/N162T, L127Q/P129A/K161S/N162G/G199H,L127Q/P129A/K161S/G199H/N200A, F156R, F156R/K161S, F156R/K161S/N162G,F156R/K161S/N162T, F156R/N162G/G199H, and F156R/G199H/N200A, wherein thepositions are numbered with reference to SEQ ID NO: 1002. In someembodiments, the polypeptide sequence of the engineeredglycosyltransferase comprises a sequence at least 90% identical to anyof SEQ ID NOS: 2648, 2650, 2652, 2654, 2656, 2658, 2660, 2662, 2664,2666, 2668, 2670, 2672, 2674, 2676, 2678, 2680, 2682, and 2684. In someembodiments, the polypeptide sequence of the engineeredglycosyltransferase comprises a sequence at least 95% identical to anyof SEQ ID NOS: 2648, 2650, 2652, 2654, 2656, 2658, 2660, 2662, 2664,2666, 2668, 2670, 2672, 2674, 2676, 2678, 2680, 2682, and 2684. In someembodiments, the polypeptide sequence of the engineeredglycosyltransferase comprises any of SEQ ID NOS: 2648, 2650, 2652, 2654,2656, 2658, 2660, 2662, 2664, 2666, 2668, 2670, 2672, 2674, 2676, 2678,2680, 2682, and 2684.

The present invention also provides engineered glycosyltransferases,wherein the polypeptide sequence of the engineered glycosyltransferasescomprise at least one mutation or mutation set at one or more positionsselected from 2/7/12/15/175/260/318, 7/12/400/435, 7/318/451,12/15/57/71/175/260/400/402, 12/15/57/220/254/260/318/402,12/15/57/318/402/435, 12/15/318/400/402/406, 12/57/175/451,12/175/260/264/318/400, 12/175/400/402/406, 12/318/402/404/406/451,12/318/402/404/451, 15/175/318/400/402, 57/175/220/260/264/402,57/175/404, 57/220/260/400/402/406, 57/260/400/402/404, 57/400/402,57/402, 152/192/195, 160/186/195, 195, 260, and 400/402, wherein thepositions are numbered with reference to SEQ ID NO: 2600. In someembodiments, the polypeptide sequence of the engineeredglycosyltransferase comprises at least one mutation or mutation set atone or more positions selected from 2-/7E/12S/15K/175S/260V/318D,7E/12S/400Q/435V, 7E/318D/451N, 12S/15K/57K/71I/175S/260V/400Q/402I,12S/15K/57K/220L/254K/260V/318D/402I, 12S/15K/57K/318D/402I/435V,12S/15K/318D/400Q/402I/406M, 12S/57K/1755/451N,12S/175S/260V/264A/318D/400Q, 12S/175S/400Q/402I/406M,12S/318D/402I/404S/406M/451N, 12S/318D/402I/404S/451N,15K/1755/318D/400Q/402I, 57K/175S/220L/260V/264A/402I, 57K/175S/404G,57K/220L/260V/400Q/402I/406M, 57K/260V/400Q/402I/404S, 57K/400Q/402I,57K/402I, 152V/192D/195P, 160V/186M/195P, 195P, 260V, and 400Q/4021,wherein the positions are numbered with reference to SEQ ID NO: 2600. Insome embodiments, the polypeptide sequence of the engineeredglycosyltransferase comprises at least one mutation or mutation set atone or more positions selected from H2-/H7E/A12S/R15K/P175S/T260V/E318D,H7E/A12S/E400Q/A435V, H7E/E318D/K451N,A12S/R15K/I57K/V71I/P175S/T260V/E400Q/L402I,A12S/R15K/I57K/C220L/T254K/T260V/E318D/L402I,A12S/R15K/I57K/E318D/L402I/A435V, A12S/R15K/E318D/E400Q/L402I/I406M,A12S/I57K/P175S/K451N, A12S/P175S/T260V/S264A/E318D/E400Q,A12S/P175S/E400Q/L402I/I406M, A12S/E318D/L402I/D404S/I406M/K451N,A12S/E318D/L402I/D404S/K451N, R15K/P175S/E318D/E400Q/L402I,I57K/P175S/C220L/T260V/S264A/L402I, I57K/P175S/D404G,I57K/C220L/T260V/E400Q/L402I/I406M, I57K/T260V/E400Q/L402I/D404S,I57K/E400Q/L402I, I57K/L402I, L152V/E192D/R195P, L160V/F186M/R195P,R195P, T260V, and E400Q/L402I, wherein the positions are numbered withreference to SEQ ID NO: 2600. In some embodiments, the polypeptidesequence of the engineered glycosyltransferase comprises at least onemutation or mutation set at one or more positions selected from 32, 135,148, 152, 186, 237, 239, 240, 323, 325, 326, 327, 330, 331, and 356,wherein the positions are numbered with reference to SEQ ID NO: 2600.

In some embodiments, the polypeptide sequence of the engineeredglycosyltransferase comprises at least one mutation or mutation set atone or more positions selected from 32R, 135A, 148A, 152V, 186V, 237T,239E, 239F, 239Y, 240A, 240P, 323L, 325G, 325R, 326M, 327V, 330A, 331C,331H, 331IS, and 356G, wherein the positions are numbered with referenceto SEQ ID NO: 2600. In some embodiments, the polypeptide sequence of theengineered glycosyltransferase comprises at least one mutation ormutation set at one or more positions selected from N32R, V135A, S148A,L152V, F186V, D237T, 1239E, 1239F, 1239Y, T240A, T240P, V323L, H325G,H325R, F326M, A327V, P330A, R331C, R331H, R331S, and F356G, wherein thepositions are numbered with reference to SEQ ID NO: 2600. In someembodiments, the polypeptide sequence of the engineeredglycosyltransferase comprises a sequence at least 90% identical to anyof SEQ ID NOS: 2686, 2688, 2690, 2692, 2694, 2696, 2698, 2700, 2702,2704, 2706, 2708, 2710, 2712, 2714, 2716, 2718, 2720, 2722, 2724, 2726,2728, 2730, 2732, 2734, 2736, 2738, 2740, 2742, 2744, 2746, 2748, 2750,2752, 2754, 2756, 2758, 2760, 2762, 2764, 2766, 2768, 2770, 2772, and2774. I n some embodiments, the polypeptide sequence of the engineeredglycosyltransferase comprises a sequence at least 95% identical to anyof SEQ ID NOS: 2686, 2688, 2690, 2692, 2694, 2696, 2698, 2700, 2702,2704, 2706, 2708, 2710, 2712, 2714, 2716, 2718, 2720, 2722, 2724, 2726,2728, 2730, 2732, 2734, 2736, 2738, 2740, 2742, 2744, 2746, 2748, 2750,2752, 2754, 2756, 2758, 2760, 2762, 2764, 2766, 2768, 2770, 2772, and2774. In some embodiments, the polypeptide sequence of the engineeredglycosyltransferase comprises any of SEQ ID NOS: 2686, 2688, 2690, 2692,2694, 2696, 2698, 2700, 2702, 2704, 2706, 2708, 2710, 2712, 2714, 2716,2718, 2720, 2722, 2724, 2726, 2728, 2730, 2732, 2734, 2736, 2738, 2740,2742, 2744, 2746, 2748, 2750, 2752, 2754, 2756, 2758, 2760, 2762, 2764,2766, 2768, 2770, 2772, and 2774.

The present invention also provides engineered glycosyltransferases,wherein the polypeptide sequence of the engineered glycosyltransferasescomprise at least one mutation or mutation set at one or more positionsselected from 14/23/274/326/329, 14/31/184/274/322/326/329/330,14/56/184/185/194/238/239/274/329/399,14/56/184/194/234/315/326/329/399/401,14/56/194/238/315/325/326/329/330/399,14/56/252/274/315/326/329/330/401, 14/184/185/194/234/325/326/329,14/184/185/194/388/399/401, 14/184/194/355/399, 14/185/194/238/399,14/185/236/238/239/274/322/326/329/355/399/401, 14/194, 14/322/326/330,14/326/330, 14/355, 14/355/399,23/31/147/184/185/238/252/325/329/330/388/401, 31/56/315/329/330, 65,65/114/132/238, 65/238/240, 147/236/238/243/315/329/330/399/401,185/194/236/239/325/326, 223/412, and 238, wherein the positions arenumbered with reference to SEQ ID NO: 2718. In some embodiments, thepolypeptide sequence of the engineered glycosyltransferase comprises atleast one mutation or mutation set at one or more positions selectedfrom 14R/23Q/274Q/326V/329A, 14R/31R/184A/274Q/322L/326V/329A/330H,14R/56K/184A/185M/194P/238M/239A/274Q/329A/399Q,14R/56K/184A/194P/234Y/315V/326V/329Q/399Q/4011,14R/56K/194P/238M/315V/325M/326V/329A/330H/399Q,14R/56K/252D/274Q/315V/326V/329A/330H/401I,14R/184A/185M/194P/234Y/325M/326V/329A,14R/184A/185M/194P/388E/399Q/401I, 14R/184A/194P/355G/399Q,14R/185M/194P/238M/399Q,14R/185M/236T/238M/239A/274Q/322L/326V/329Q/355G/399Q/401I, 14R/194P,14R/322L/326V/330H, 14R/326V/330H, 14R/355G, 14R/355G/399Q,23Q/31R/147A/184A/185M/238M/252D/325M/329A/330H/388E/4011,31R/56K/315V/329A/330H, 65D, 65D/114E/132R/238M, 65D/238T/240S,147A/236T/238M/243G/315V/329A/330H/399Q/401I,185M/194P/236T/239A/325M/326V, 223T/412, and 238M, wherein the positionare numbered with reference to SEQ ID NO: 2718. In some embodiments, thepolypeptide sequence of the engineered glycosyltransferase comprises atleast one mutation or mutation set at one or more positions selectedfrom K14R/L23Q/D274Q/A326V/P329A,K14R/N31R/M184A/D274Q/V322L/A326V/P329A/R330H,K14R/I56K/M184A/F185M/R194P/I238M/T239A/D274Q/P329A/E399Q,K14R/I56K/M184A/R194P/F234Y/I315V/A326V/P329Q/E399Q/L401I,K14R/I56K/R194P/I238M/I315V/F325M/A326V/P329A/R330H/E399Q,K14R/I56K/G252D/D274Q/I315V/A326V/P329A/R330H/L401I,K14R/M184A/F185M/R194P/F234Y/F325M/A326V/P329A,K14R/M184A/F185M/R194P/D388E/E399Q/L401I, K14R/M184A/R194P/F355G/E399Q,K14R/F185M/R194P/I238M/E399Q,K14R/F185M/D236T/I238M/T239A/D274Q/V322L/A326V/P329Q/F355G/E399Q/L401I,K14R/R194P, K14R/V322L/A326V/R330H, K14R/A326V/R330H, K14R/F355G,K14R/F355G/E399Q,L23Q/N31R/S147A/M184A/F185M/I238M/G252D/F325M/P329A/R330H/D388E/L401I,N31R/I56K/I315V/P329A/R330H, E65D, E65D/Q114E/H132R/I238M,E65D/I238T/N240S, S147A/D236T/I238M/D243G/I315V/P329A/R330H/E399Q/L401I,F185M/R194P/D236T/T239A/F325M/A326V, S223T/I412S, and I238M, wherein thepositions are numbered with reference to SEQ ID NO: 2718. In someembodiments, the polypeptide sequence of the engineeredglycosyltransferase comprises at least one mutation or mutation set atone or more positions selected from 11, 45, 55, 56, 58, 65, 104, 113,114, 132, 135, 138, 165, 238, 256, 273, 286, 309, 391, 422, 430, and449, wherein the positions are numbered with reference to SEQ ID NO:2718.

In some embodiments, the polypeptide sequence of the engineeredglycosyltransferase comprises at least one mutation or mutation set atone or more positions selected from 11G, 11Q, 45F, 45V, 55L, 56T, 58R,65N, 65S, 104L, 113V, 114R, 132Q, 132S, 135L, 138G, 138K, 165P, 238G,256P, 273R, 286R, 309E, 309H, 391R, 422R, 430L, 430V, and 449F, whereinthe positions are numbered with reference to SEQ ID NO: 2718. In someembodiments, the polypeptide sequence of the engineeredglycosyltransferase comprises at least one mutation or mutation set atone or more positions selected from S11G, S11Q, L45F, L45V, I55L, I56T,K58R, E65N, E65S, M104L, L113V, Q114R, H132Q, H132S, N135L, N138G,N138K, E165P, 1238G, E256P, E273R, N286R, K309E, K309H, N391R, K422R,E430L, E430V, and Y449F, wherein the positions are numbered withreference to SEQ ID NO: 2718. In some embodiments, the polypeptidesequence of the engineered glycosyltransferase comprises a sequence atleast 90% identical to any of SEQ ID NOS: 2776, 2778, 2780, 2782, 2784,2786, 2788, 2790, 2792, 2794, 2796, 2798, 2800, 2802, 2804, 2806, 2808,2810, 2812, 2814, 2816, 2818, 2820, 2822, 2824, 2826, 2828, 2830, 2832,2834, 2836, 2838, 2840, 2842, 2844, 2846, 2848, 2850, 2852, 2854, 2856,2858, 2860, 2862, 2864, 2866, 2868, 2870, 2872, 2874, 2876, 2878, 2880,and 2882. In some embodiments, the polypeptide sequence of theengineered glycosyltransferase comprises a sequence at least 95%identical to any of SEQ ID NOS: 2776, 2778, 2780, 2782, 2784, 2786,2788, 2790, 2792, 2794, 2796, 2798, 2800, 2802, 2804, 2806, 2808, 2810,2812, 2814, 2816, 2818, 2820, 2822, 2824, 2826, 2828, 2830, 2832, 2834,2836, 2838, 2840, 2842, 2844, 2846, 2848, 2850, 2852, 2854, 2856, 2858,2860, 2862, 2864, 2866, 2868, 2870, 2872, 2874, 2876, 2878, 2880, and2882. In some embodiments, the polypeptide sequence of the engineeredglycosyltransferase comprises any of SEQ ID NOS: 2776, 2778, 2780, 2782,2784, 2786, 2788, 2790, 2792, 2794, 2796, 2798, 2800, 2802, 2804, 2806,2808, 2810, 2812, 2814, 2816, 2818, 2820, 2822, 2824, 2826, 2828, 2830,2832, 2834, 2836, 2838, 2840, 2842, 2844, 2846, 2848, 2850, 2852, 2854,2856, 2858, 2860, 2862, 2864, 2866, 2868, 2870, 2872, 2874, 2876, 2878,2880, and 2882.

The present invention also provides engineered glycosyltransferases,wherein the polypeptide sequence of the engineered glycosyltransferasescomprise at least one mutation or mutation set at one or more positionsselected from 23/31/185/324/401, 23/31/185/355,31/134/185/252/274/324/388, 31/134/238/252/322/324/388, 31/134/252/324,31/184/185/238/239/322, 31/236, 31/322, 31/388,134/184/185/234/236/239/274/324/388, 184/185/322,234/236/238/322/324/355, 236, 238/324/329/355, 322/324, and324/329/355/401, numbered with reference to SEQ ID NO: 2814. In someembodiments, the polypeptide sequence of the engineeredglycosyltransferase comprises at least one mutation or mutation set atone or more positions selected from 23Q/31R/85M/324G/401I,23Q/31R/185M/355G, 31R/134A/185M/252D/274Q/324G/388E,31R/134A/238E/252D/322L/324G/388E, 31R/134A/252D/324G,31R/184A/185M/238Y/239A/322L, 31R/236T, 31R/322L, 31R/388E,134A/184A/185M/234Y/236T/239A/274Q/324G/388E, 184A/185M/322L,234Y/236T/238E/322L/324G/355G, 236T, 238Y/324G/329Q/355G, 322L/324G, and324G/329Q/355G/401I, numbered with reference to SEQ ID NO: 2814. In someembodiments, the polypeptide sequence of the engineeredglycosyltransferase comprises at least one mutation or mutation set atone or more positions selected from L23Q/N31R/F185M/H324G/L401I,L23Q/N31R/F185M/F355G, N31R/V134A/F185M/G252D/D274Q/H324G/D388E,N31R/V134A/M238E/G252D/V322L/H324G/D388E, N31R/V134A/G252D/H324G,N31R/M184A/F185M/M238Y/T239A/V322L, N31R/D236T, N31R/V322L, N31R/D388E,V134A/M184A/F185M/F234Y/D236T/T239A/D274Q/H324G/D388E,M184A/F185M/V322L, F234Y/D236T/M238E/V322L/H324G/F355G, D236T,M238Y/H324G/A329Q/F355G, V322L/H324G, and H324G/A329Q/F355G/L401I,numbered with reference to SEQ ID NO: 2814. In some embodiments, thepolypeptide sequence of the engineered glycosyltransferase comprises atleast one mutation or mutation set at one or more positions selectedfrom 122, 164, 176, 177, 316, 325, 400, 425, 426, 427, 440, and 446,numbered with reference to SEQ ID NO: 2814. In some embodiments, thepolypeptide sequence of the engineered glycosyltransferase comprises atleast one mutation or mutation set at one or more positions selectedfrom 122L, 164H, 164M, 164R, 176K, 176L, 176N, 176R, 177A, 316R, 325L,400V, 425R, 426A, 426R, 427R, 440R, and 446R, numbered with reference toSEQ ID NO: 2814. In some embodiments, the polypeptide sequence of theengineered glycosyltransferase comprises at least one mutation ormutation set at one or more positions selected from I122L, V164H, V164M,V164R, V176K, V176L, V176N, V176R, E177A, G316R, M325L, T400V, K425R,S426A, S426R, I427R, I440R, and S446R, numbered with reference to SEQ IDNO: 2814. In some embodiments, the polypeptide sequence of theengineered glycosyltransferase comprises a sequence at least 90%identical to any of SEQ ID NOS: 2884, 2886, 2888, 2890, 2892, 2894,2896, 2898, 2900, 2902, 2904, 2906, 2908, 2910, 2912, 2914, 2916, 2918,2920, 2922, 2924, 2926, 2928, 2830, 2932, 2934, 2936, 2938, 2940, 2942,2944, 2946, 2948, and 2950, numbered with reference to SEQ ID NO: 2814.In some embodiments, the polypeptide sequence of the engineeredglycosyltransferase comprises a sequence at least 95% identical to anyof SEQ ID NOS: 2884, 2886, 2888, 2890, 2892, 2894, 2896, 2898, 2900,2902, 2904, 2906, 2908, 2910, 2912, 2914, 2916, 2918, 2920, 2922, 2924,2926, 2928, 2830, 2932, 2934, 2936, 2938, 2940, 2942, 2944, 2946, 2948,and 2950. In some embodiments, the polypeptide sequence of theengineered glycosyltransferase comprises any of SEQ ID NOS: 2884, 2886,2888, 2890, 2892, 2894, 2896, 2898, 2900, 2902, 2904, 2906, 2908, 2910,2912, 2914, 2916, 2918, 2920, 2922, 2924, 2926, 2928, 2830, 2932, 2934,2936, 2938, 2940, 2942, 2944, 2946, 2948, and 2950.

The present invention also provides engineered glycosyltransferases,wherein the polypeptide sequence of the engineered glycosyltransferasescomprise at least one mutation or mutation set at one or more positionsselected from 11, 11/45/58/132/138/286, 11/58, 11/58/65/104/107/138/286,11/58/65/107/135/223/286/391/430, 11/58/65/135/138,11/58/104/107/114/138/223/391, 11/58/114/286/309/391,11/58/132/165/286/391/422/430, 11/58/138/309,11/65/104/107/138/165/286/391/430, 11/65/107/135/165/391/430,11/65/132/135/138/223/391, 11/65/132/135/252, 11/65/132/391/430,11/65/135/138, 11/104/132/138/309/391, 11/104/132/138/391,11/107/114/223/309/430, 11/107/138, 11/114/135/138/223,11/114/223/252/286/391, 11/132/135/138/223/286, 11/132/138/223/286/391,11/138/165/223/309, 58/65/138, 58/65/138/165, 58/65/165/309/430,58/104/114/165/391, 107/114/132/138, 107/430, 135/138/165/309/430, and223/309, wherein the positions are numbered with reference to SEQ ID NO:2884. In some embodiments, the polypeptide sequence of the engineeredglycosyltransferase comprises at least one mutation or mutation set atone or more positions selected from 11G/45V/58R/132Q/138G/286R,11G/58R/104L/107G/114R/138K/223T/391R, 11G/58R/114R/286R/309H/391R,11G/58R/132Q/165P/286R/391R/422S/430L, 11G/65N/132Q/391R/430L,11G/104L/132Q/138G/309H/391R, 11G/107G/114R/223T/309H/430L, 11Q,11Q/58R, 11Q/58R/65N/104L/107G/138G/286R,11Q/58R/65N/107G/135L/223T/286R/391R/430L, 11Q/58R/65N/135L/138G,11Q/58R/138G/309E, 11Q/65N/104L/107G/138G/165P/286R/391R/430L,11Q/65N/107G/135L/165P/391R/430L, 11Q/65N/132Q/135L/138G/223T/391R,11Q/65N/132Q/135L/252D, 11Q/65N/135L/138G, 11Q/104L/132Q/138K/391R,11Q/107G/138G, 11Q/114R/135L/138G/223T, 11Q/114R/223T/252D/286R/391R,11Q/132Q/135L/138K/223T/286R, 11Q/132Q/138G/223T/286R/391R,11Q/138K/165P/223T/309H, 58R/65N/138G/165P, 58R/65N/138K,58R/65N/165P/309H/430L, 58R/104L/114R/165P/391R, 107G/114R/132Q/138G,107G/430L, 135L/138G/165P/309H/430L, and 223T/309E, wherein thepositions are numbered with reference to SEQ ID NO: 2884. In someembodiments, the polypeptide sequence of the engineeredglycosyltransferase comprises at least one mutation or mutation set atone or more positions selected from SI1G/L45V/K58R/H132Q/N138G/N286R,S11G/K58R/M104L/P107G/Q114R/N138K/S223T/N391R,S11G/K58R/Q114R/N286R/K309H/N391R,S11G/K58R/H132Q/E165P/N286R/N391R/K422S/E430L,S11G/E65N/H132Q/N391R/E430L, S11G/M104L/H132Q/N138G/K309H/N391R,S11G/P107G/Q114R/S223T/K309H/E430L, S11Q, S11Q/K58R,S11Q/K58R/E65N/M104L/P107G/N138G/N286R,S11Q/K58R/E65N/P107G/N135L/S223T/N286R/N391R/E430L,S11Q/K58R/E65N/N135L/N138G, S11Q/K58R/N138G/K309E,S11Q/E65N/M104L/P107G/N138G/E165P/N286R/N391R/E430L,S11Q/E65N/P107G/N135L/E165P/N391R/E430L,S11Q/E65N/H132Q/N135L/N138G/S223T/N391R, S11Q/E65N/H132Q/N135L/G252D,S11Q/E65N/N135L/N138G, S11Q/M104L/H132Q/N138K/N391R, S11Q/P107G/N138G,S11Q/Q114R/N135L/N138G/S223T, S11Q/Q114R/S223T/G252D/N286R/N391R,S11Q/H132Q/N135L/N138K/S223T/N286R, S11Q/H132Q/N138G/S223T/N286R/N391R,S11Q/N138K/E165P/S223T/K309H, K58R/E65N/N138G/E165P, K58R/E65N/N138K,K58R/E65N/E165P/K309H/E430L, K58R/M104L/Q114R/E165P/N391R,P107G/Q114R/H132Q/N138G, P107G/E430L, N135L/N138G/E165P/K309H/E430L, andS223T/K309E, wherein the positions are numbered with reference to SEQ IDNO: 2884. In some embodiments, the polypeptide sequence of theengineered glycosyltransferase comprises a sequence at least 90%identical to any of SEQ ID NOS: 2952, 2954, 2956, 2958, 2960, 2962,2964, 2966, 2968, 2970, 2972, 2974, 2976, 2978, 2980, 2982, 2984, 2986,2988, 2990, 2992, 2994, 2996, 2998, 3000, 3002, 3004, 3006, 3008, 3010,3012, 3014, and 3016. In some embodiments, the polypeptide sequence ofthe engineered glycosyltransferase comprises a sequence at least 95%identical to any of SEQ ID NOS: 2952, 2954, 2956, 2958, 2960, 2962,2964, 2966, 2968, 2970, 2972, 2974, 2976, 2978, 2980, 2982, 2984, 2986,2988, 2990, 2992, 2994, 2996, 2998, 3000, 3002, 3004, 3006, 3008, 3010,3012, 3014, and 3016. In some embodiments, the polypeptide sequence ofthe engineered glycosyltransferase comprises any of SEQ ID NOS: 2952,2954, 2956, 2958, 2960, 2962, 2964, 2966, 2968, 2970, 2972, 2974, 2976,2978, 2980, 2982, 2984, 2986, 2988, 2990, 2992, 2994, 2996, 2998, 3000,3002, 3004, 3006, 3008, 3010, 3012, 3014, and 3016.

The present invention also provides engineered glycosyltransferases,wherein the polypeptide sequence of the engineered glycosyltransferasescomprise at least one mutation or mutation set at one or more positionsselected from 8/448, 58/107/122/176/236/324/325/400/426/427,58/107/122/236/324/400/425/446, 58/107/122/322/400/425/427/440/446,58/107/164/400, 58/107/236/400, 58/107/400, 58/122/164/236/446,58/122/176/236/400/446, 58/122/176/322/324/426/427, 58/122/322/325,58/122/325, 58/122/440, 58/164/176, 58/164/324/425/427, 58/176/236,58/236, 107/122/236/425/426/446, 107/164/236/400/446,107/176/322/325/440/446, 107/176/400/425/427/440, 107/236, 107/440,122/164/176/324/400, 122/164/400/440, 122/164/400/440/446,122/176/236/400, 122/176/324/400/440, 122/400, 122/425/426/446,122/440/446, 139, 139/252, 164, 164/271/425/426,164/322/324/325/400/425/440/446, 164/400, 176/400, 189, and324/400/425/426/440/446, wherein the positions are numbered withreference to SEQ ID NO: 3016. In some embodiments, the polypeptidesequence of the engineered glycosyltransferase comprises at least onemutation or mutation set at one or more positions selected from 8S/448A,58R/107G/122L/176R/236T/324G/325L/400V/426R/427R,58R/107G/122L/236T/324G/400V/425R/446R,58R/107G/122L/322L/400V/425R/427R/440R/446R, 58R/107G/164H/400V,58R/107G/236T/400V, 58R/107G/400V, 58R/122L/164H/236T/446R,58R/122L/176R/236T/400V/446R, 58R/122L/176R/322L/324G/426A/427R,58R/122L/322L/325L, 58R/122L/325L, 58R/122L/440R, 58R/164H/176R,58R/164H/324G/425R/427R, 58R/176R/236T, 58R/236T,107G/122L/236T/425R/426A/446R, 107G/164H/236T/400V/446R,107G/176R/322L/325L/440R/446R, 107G/176R/400V/425R/427R/440R, 107G/236T,107G/440R, 122L/164H/176R/324G/400V, 122L/164H/400V/440R,122L/164H/400V/440R/446R, 122L/176R/236T/400V, 122L/176R/324G/400V/440R,122L/400V, 122L/425R/426R/446R, 122L/440R/446R, 139V, 139V/252D, 164H,164H/271G/425R/426R, 164H/322L/324G/325L/400V/425R/440R/446R, 164H/400V,176R/400V, 189R, and 324G/400V/425R/426R/440R/446R, wherein thepositions are numbered with reference to SEQ ID NO: 3016. In someembodiments, the polypeptide sequence of the engineeredglycosyltransferase comprises at least one mutation or mutation set atone or more positions selected from G8S/K448A,K58R/P107G/I122L/V176R/D236T/H324G/M325L/T400V/S426R/I427R,K58R/P107G/I122L/D236T/H324G/T400V/K425R/S446R,K58R/P107G/I122L/V322L/T400V/K425R/I427R/I440R/S446R,K58R/P107G/V164H/T400V, K58R/P107G/D236T/T400V, K58R/P107G/T400V,K58R/I122L/V164H/D236T/S446R, K58R/I122L/V176R/D236T/T400V/S446R,K58R/I122L/V176R/V322L/H324G/S426A/I427R, K58R/I122L/V322L/M325L,K58R/I122L/M325L, K58R/I122L/I440R, K58R/V164H/V176R,K58R/V164H/H324G/K425R/I427R, K58R/V176R/D236T, K58R/D236T,P107G/I122L/D236T/K425R/S426A/S446R, P107G/V164H/D236T/T400V/S446R,P107G/V176R/V322L/M325L/I440R/S446R,P107G/V176R/T400V/K425R/I427R/I440R, P107G/D236T, P107G/I440R,I122L/V164H/V176R/H324G/T400V, I122L/V164H/T400V/I440R,I122L/V164H/T400V/I440R/S446R, I122L/V176R/D236T/T400V,I122L/V176R/H324G/T400V/I440R, I122L/T400V, I122L/K425R/S426R/S446R,I122L/I440R/S446R, I139V, I139V/G252D, V164H, V164H/S271G/K425R/S426R,V164H/V322L/H324G/M325L/T400V/K425R/I440R/S446R, V164H/T400V,V176R/T400V, P189R, and H324G/T400V/K425R/S426R/I440R/S446R, wherein thepositions are numbered with reference to SEQ ID NO: 3016. In someembodiments, the polypeptide sequence of the engineeredglycosyltransferase comprises at least one mutation or mutation set atone or more positions selected from 9, 65, 106, 115, 116, 172, 178, 200,210, 213, 240, 242, 245, 255, 324/423, 385, 408, 409, 411, 412, 415,416, 423, and 447, wherein the positions are numbered with reference toSEQ ID NO: 3016.

In some embodiments, the polypeptide sequence of the engineeredglycosyltransferase comprises at least one mutation or mutation set atone or more positions selected from 9C, 9M, 655, 106A, 115D, 115R, 116V,172R, 172S, 178K, 200A, 200T, 200V, 210L, 210V, 213G, 240C, 240E, 240L,240P, 240V, 242I, 242Y, 245M, 255P, 324R/423R, 385R, 408P, 409L, 411Q,412R, 415A, 415H, 415K, 415R, 416R, 423A, 423R, 447L, and 447R, whereinthe positions are numbered with reference to SEQ ID NO: 3016. In someembodiments, the polypeptide sequence of the engineeredglycosyltransferase comprises at least one mutation or mutation set atone or more positions selected from S9C, S9M, N65S, K106A, N115D, N115R,L116V, N172R, N172S, Q178K, G200A, G200T, G200V, E210L, E210V, A213G,N240C, N240E, N240L, N240P, N240V, A242I, A242Y, K245M, D255P,H324R/N423R, V385R, E408P, T409L, E411Q, I412R, G415A, G415H, G415K,G415R, K416R, N423A, N423R, N447L, and N447R, wherein the positions arenumbered with reference to SEQ ID NO: 3016. In some embodiments, thepolypeptide sequence of the engineered glycosyltransferase comprises asequence at least 90% identical to any of SEQ ID NOS: 3018, 3020, 3022,3024, 3026, 3028, 3030, 3032, 3034, 3036, 3038, 3040, 3042, 3044, 3046,3048, 3050, 3052, 3054, 3056, 3058, 3060, 3062, 3064, 3066, 3068, 3070,3072, 3074, 3076, 3078, 3080, 3082, 3084, 3086, 3088, 3090, 3092, 3094,3096, 3098, 3100, 3102, 3104, 3106, 3108, 3110, 3112, 3114, 3116, 3118,3120, 3122, 3124, 3126, 3128, 3130, 3132, 3134, 3136, 3138, 3140, 3142,3144, 3146, 3148, 3150, 3152, 3154, 3156, 3158, 3160, 3162, 3164, 3166,3168, 3170, 3172, 3174, 3176, 3178, and 3180. In some embodiments, thepolypeptide sequence of the engineered glycosyltransferase comprises asequence at least 95% identical to any of SEQ ID NOS: 3018, 3020, 3022,3024, 3026, 3028, 3030, 3032, 3034, 3036, 3038, 3040, 3042, 3044, 3046,3048, 3050, 3052, 3054, 3056, 3058, 3060, 3062, 3064, 3066, 3068, 3070,3072, 3074, 3076, 3078, 3080, 3082, 3084, 3086, 3088, 3090, 3092, 3094,3096, 3098, 3100, 3102, 3104, 3106, 3108, 3110, 3112, 3114, 3116, 3118,3120, 3122, 3124, 3126, 3128, 3130, 3132, 3134, 3136, 3138, 3140, 3142,3144, 3146, 3148, 3150, 3152, 3154, 3156, 3158, 3160, 3162, 3164, 3166,3168, 3170, 3172, 3174, 3176, 3178, and 3180. In some embodiments, thepolypeptide sequence of the engineered glycosyltransferase comprises anyof SEQ ID NOS: 3018, 3020, 3022, 3024, 3026, 3028, 3030, 3032, 3034,3036, 3038, 3040, 3042, 3044, 3046, 3048, 3050, 3052, 3054, 3056, 3058,3060, 3062, 3064, 3066, 3068, 3070, 3072, 3074, 3076, 3078, 3080, 3082,3084, 3086, 3088, 3090, 3092, 3094, 3096, 3098, 3100, 3102, 3104, 3106,3108, 3110, 3112, 3114, 3116, 3118, 3120, 3122, 3124, 3126, 3128, 3130,3132, 3134, 3136, 3138, 3140, 3142, 3144, 3146, 3148, 3150, 3152, 3154,3156, 3158, 3160, 3162, 3164, 3166, 3168, 3170, 3172, 3174, 3176, 3178,and 3180.

The present invention also provides engineered glycosyltransferases,wherein the polypeptide sequence of the engineered glycosyltransferasescomprise at least one mutation or mutation set at one or more positionsselected from, 8, 8/107/139/164/415/416/440/448,8/107/139/255/322/325/415/416/440/448, 8/107/164/255, 8/107/164/423/440,8/139, 8/139/164/200/236/240/322/440/448, 8/139/164/236/416/423/440,8/139/189/240/325/416, 8/139/200/236/240/255/423, 8/139/255/415/416,8/164, 8/164/189/200/322/324/325/416/448, 8/164/240/423,8/164/252/255/448, 8/164/448, 8/236/240/252/448, 8/240/423,8/252/255/322/325/448, 12/164/440, 107/139,107/139/200/240/322/324/325/448, 107/236/240/325/440,107/240/252/423/448, 107/423, 139, 139/164/236/240, 139/255/325/415/440,164/189, 164/189/236/240, 164/189/240/252/415/423, 164/200/236,164/200/236/240/324/416/440, 164/200/236/255/322/324/423/440, 164/236,164/236/240/440, 164/236/423, 164/322/325, 164/322/325/416/423,164/416/448, 200/236/322/325/416, 200/322/325/415/448, 236/415/416, 240,240/252/255/322/415/416, 240/252/255/415/448, 255/423, 325, and415/416/448, wherein the positions are numbered with reference to SEQ IDNO: 3082. In some embodiments, the polypeptide sequence of theengineered glycosyltransferase comprises at least one mutation ormutation set at one or more positions selected from 2-, 8S,8S/107G/139V/164H/415A/416R/440R/448A,8S/107G/139V/255P/322L/325L/415A/416R/440R/448A, 8S/107G/164H/255P,8S/107G/164H/423R/440R, 8S/139V,8S/139V/164H/200A/236T/240E/322L/440R/448A,8S/139V/164H/236T/416R/423R/440R, 8S/139V/189R/240E/325L/416R,8S/139V/200A/236T/240E/255P/423R, 8S/139V/255P/415A/416R, 8S/164H,8S/164H/189R/200A/322L/324G/325L/416R/448A, 8S/164H/240E/423R,8S/164H/252D/255P/448A, 8S/164H/448A, 8S/236T/240E/252D/448A,8S/240E/423R, 8S/252D/255P/322L/325L/448A, 12S/164H/440R, 107G/139V,107G/139V/200A/240E/322L/324G/325L/448A, 107G/236T/240E/325L/440R,107G/240E/252D/423R/448A, 107G/423R, 139V, 139V/164H/236T/240E,139V/255P/325L/415A/440R, 164H/189R, 164H/189R/236T/240E,164H/189R/240E/252D/415A/423R, 164H/200A/236T,164H/200A/236T/240E/324G/416R/440R,164H/200A/236T/255P/322L/324G/423R/440R, 164H/236T, 164H/236T/240E/440R,164H/236T/423R, 164H/322L/325L, 164H/322L/325L/416R/423R,164H/416R/448A, 200A/236T/322L/325L/416R, 200A/322L/325L/415A/448A,236T/415A/416R, 240E, 240E/252D/255P/322L/415A/416R,240E/252D/255P/415A/448A, 255P/423R, 325L, and 415A/416R/448A, whereinthe positions are numbered with reference to SEQ ID NO: 3082. In someembodiments, the polypeptide sequence of the engineeredglycosyltransferase comprises at least one mutation or mutation set atone or more positions selected from H2-, G8S,G8S/P107G/I139V/V164H/G415A/K416R/I440R/K448A,G8S/P107G/I139V/D255P/V322L/M325L/G415A/K416R/I440R/K448A,G8S/P107G/V164H/D255P, G8S/P107G/V164H/N423R/I440R, G8S/I139V,G8S/I139V/V164H/G200A/D236T/N240E/V322L/I440R/K448A,G8S/I139V/V164H/D236T/K416R/N423R/I440R,G8S/I139V/P189R/N240E/M325L/K416R,G8S/I139V/G200A/D236T/N240E/D255P/N423R, G8S/I139V/D255P/G415A/K416R,G8S/V164H, G8S/V164H/P189R/G200A/V322L/H324G/M325L/K416R/K448A,G8S/V164H/N240E/N423R, G8S/V164H/G252D/D255P/K448A, G8S/V164H/K448A,G8S/D236T/N240E/G252D/K448A, G8S/N240E/N423R,G8S/G252D/D255P/V322L/M325L/K448A, T12S/V164H/I440R, P107G/I139V,P107G/I139V/G200A/N240E/V322L/H324G/M325L/K448A,P107G/D236T/N240E/M325L/I440R, P107G/N240E/G252D/N423R/K448A,P107G/N423R, I139V, I139V/V164H/D236T/N240E,I139V/D255P/M325L/G415A/I440R, V164H/P189R, V164H/P189R/D236T/N240E,V164H/P189R/N240E/G252D/G415A/N423R, V164H/G200A/D236T,V164H/G200A/D236T/N240E/H324G/K416R/I440R,V164H/G200A/D236T/D255P/V322L/H324G/N423R/I440R, V164H/D236T,V164H/D236T/N240E/I440R, V164H/D236T/N423R, V164H/V322L/M325L,V164H/V322L/M325L/K416R/N423R, V164H/K416R/K448A,G200A/D236T/V322L/M325L/K416R, G200A/V322L/M325L/G415A/K448A,D236T/G415A/K416R, N240E, N240E/G252D/D255P/V322L/G415A/K416R,N240E/G252D/D255P/G415A/K448A, D255P/N423R, M325L, andG415A/K416R/K448A, wherein the positions are numbered with reference toSEQ ID NO: 3082. In some embodiments, the polypeptide sequence of theengineered glycosyltransferase comprises at least one mutation ormutation set at one or more positions selected from 2, 3, 8, 34, 72, 73,75, 113, 114, 186, 189, 221, 235, 237, 239, 256, 286, 299, 305, 309,312, 313, 323, 355, 389, 406, 422, 438, and 446, wherein the positionsare numbered with reference to SEQ ID NO: 3082. In some embodiments, thepolypeptide sequence of the engineered glycosyltransferase comprises atleast one mutation or mutation set at one or more positions selectedfrom 2N, 2S, 2T, 31, 8S, 34R, 72Y, 73A, 73P, 73T, 73V, 75H, 113, 114V,186G, 186I, 189S, 221K, 235M, 237L, 237M, 237V, 239A, 256I, 256L, 2565,256T, 286L, 286S, 299A, 299L, 299R, 299V, 305G, 309R, 312S, 312T, 312V,313D, 323P, 355A, 389F, 389G, 406F, 406G, 406N, 406Q, 422C, 422R, 4225,438T, 446H, and 446P, wherein the positions are numbered with referenceto SEQ ID NO: 3082. In some embodiments, the polypeptide sequence of theengineered glycosyltransferase comprises at least one mutation ormutation set at one or more positions selected from H2N, H2S, H2T, H3I,G8S, K34R, L72Y, H73A, H73P, H73T, H73V, P75H, L113I, Q114V, E186G,E186I, P189S, E221K, Q235M, P237L, P237M, P237V, T239A, E256I, E256L,E256S, E256T, N286L, N286S, E299A, E299L, E299R, E299V, D305G, K309R,L312S, L312T, L312V, E313D, D323P, F355A, D389F, D389G, T406F, T406G,T406N, T406Q, K422C, K422R, K422S, E438T, R446H, and R446P, wherein thepositions are numbered with reference to SEQ ID NO: 3082. In someembodiments, the polypeptide sequence of the engineeredglycosyltransferase comprises at least one mutation or mutation set atone or more positions selected from 20/126/128/155/161,20/126/128/160/161, 20/126/128/160/161/198, 20/126/128/161,20/126/128/161/198/199, 20/126/155/160/198, 20/126/155/161, 20/126/160,20/126/160/161, 20/126/161, 20/128/155/160/161/198, 20/128/155/161/199,20/128/155/199, 20/128/160/161/198, 20/155/160, 20/155/160/161,20/155/161, 20/155/161/199, 20/160/161/198, 121/126/128/161/369,126/128/155/160/161, 126/128/155/160/161/199, 126/128/155/161/199,126/128/160/161/369, 126/128/160/198/369, 126/128/160/199/369,126/128/160/369, 126/128/161/199/369, 126/128/161/369, 126/128/369,126/160/161/199/369, 126/160/198/369, 126/196/198/369, 126/198/369,126/199/369, 126/369, 128/155/160/161, 128/155/160/161/199,128/155/160/198, 128/155/199, 128/160/161/369, 128/161/199/369,128/198/199/369, 128/199/369, 128/369, 155/161/198/199, 155/199,160/161/369, 161/198/369, 161/369, and 199/369, wherein the positionsare numbered with reference to SEQ ID NO: 3082. In some embodiments, thepolypeptide sequence of the engineered glycosyltransferase comprises atleast one mutation or mutation set at one or more positions selectedfrom 20Y/126H/128A/155R/161T, 20Y/126H/128A/161T,20Y/126H/155R/160S/198P, 20Y/126H/160S, 20Y/126H/160S/161T,20Y/126Q/128A/160S/161T, 20Y/126Q/128A/160S/161T/198H,20Y/126Q/128A/161T/198H/199A, 20Y/126Q/155R/161G, 20Y/126Q/161G,20Y/128A/155R/160S/161T/198H, 20Y/128A/155R/161T/199A,20Y/128A/155R/199A, 20Y/128A/160S/161T/198P, 20Y/155R/160S,20Y/155R/160S/161G, 20Y/155R/161T, 20Y/155R/161T/199A,20Y/160S/161T/198H, 121F/126H/128A/161T/369N, 126H/128A/155R/160S/161T,126H/128A/160S/198H/369N, 126H/128A/161G/199A/369N, 126H/128A/161G/369N,126H/128A/369N, 126H/160S/161G/199A/369N, 126H/160S/198P/369N,126H/196V/198P/369N, 126Q/128A/155R/160S/161T/199A,126Q/128A/155R/161G/199A, 126Q/128A/160S/161T/369N,126Q/128A/160S/199A/369N, 126Q/128A/160S/369N, 126Q/198P/369N,126Q/199A/369N, 126Q/369N, 128A/155R/160S/161T,128A/155R/160S/161T/199A, 128A/155R/160S/198P, 128A/155R/199A,128A/160S/161G/369N, 128A/161G/199A/369N, 128A/198H/199A/369N,128A/199A/369N, 128A/369N, 155R/161G/198H/199A, 155R/199A,160S/161G/369N, 160S/161T/369N, 161T/198P/369N, 161T/369N, and199A/369N, wherein the positions are numbered with reference to SEQ IDNO: 3082. In some embodiments, the polypeptide sequence of theengineered glycosyltransferase comprises at least one mutation ormutation set at one or more positions selected fromW20Y/L126H/P128A/F155R/R161T, W20Y/L126H/P128A/R161T,W20Y/L126H/F155R/K160S/G198P, W20Y/L126H/K160S, W20Y/L126H/K160S/R161T,W20Y/L126Q/P128A/K160S/R161T, W20Y/L126Q/P128A/K160S/R161T/G198H,W20Y/L126Q/P128A/R161T/G198H/N199A, W20Y/L126Q/F155R/R161G,W20Y/L126Q/R161G, W20Y/P128A/F155R/K160S/R161T/G198H,W20Y/P128A/F155R/R161T/N199A, W20Y/P128A/F155R/N199A,W20Y/P128A/K160S/R161T/G198P, W20Y/F155R/K160S, W20Y/F155R/K160S/R161G,W20Y/F155R/R161T, W20Y/F155R/R161T/N199A, W20Y/K160S/R161T/G198H,V121F/L126H/P128A/R161T/P369N, L126H/P128A/F155R/K160S/R161T,L126H/P128A/K160S/G198H/P369N, L126H/P128A/R161G/N199A/P369N,L126H/P128A/R161G/P369N, L126H/P128A/P369N,L126H/K160S/R161G/N199A/P369N, L126H/K160S/G198P/P369N,L126H/A196V/G198P/P369N, L126Q/P128A/F155R/K160S/R161T/N199A,L126Q/P128A/F155R/R161G/N199A, L126Q/P128A/K160S/R161T/P369N,L126Q/P128A/K160S/N199A/P369N, L126Q/P128A/K160S/P369N,L126Q/G198P/P369N, L126Q/N199A/P369N, L126Q/P369N,P128A/F155R/K160S/R161T, P128A/F155R/K160S/R161T/N199A,P128A/F155R/K160S/G198P, P128A/F155R/N199A, P128A/K160S/R161G/P369N,P128A/R161G/N199A/P369N, P128A/G198H/N199A/P369N, P128A/N199A/P369N,P128A/P369N, F155R/R161G/G198H/N199A, F155R/N199A, K160S/R161G/P369N,K160S/R161T/P369N, R161T/G198P/P369N, R161T/P369N, and N199A/P369N,wherein the positions are numbered with reference to SEQ ID NO: 3082. Insome embodiments, the polypeptide sequence of the engineeredglycosyltransferase comprises a sequence at least 90% identical to anyof SEQ ID NOS: 3182, 3184, 3186, 3188, 3190, 3192, 3194, 3196, 3198,3200, 3202, 3204, 3206, 3208, 3210, 3212, 3214, 3216, 3218, 3220, 3222,3224, 3226, 3228, 3230, 3232, 3234, 3236, 3238, 3240, 3242, 3244, 3246,3248, 3250, 3252, 3254, 3256, 3258, 3260, 3262, 3264, 3266, 3268, 3270,3272, 3274, 3276, 3278, 3280, 3282, 3284, 3286, 3288, 3290, 3292, 3294,3296, 3298, 3300, 3302, 3304, 3306, 3308, 3310, 3312, 3314, 3316, 3318,3320, 3322, 3324, 3326, 3328, 3330, 3332, 3334, 3336, 3338, 3340, 3342,3344, 3346, 3348, 3350, 3352, 3354, 3356, 3358, 3360, 3362, 3364, 3366,3368, 3370, 3372, 3374, 3376, 3378, 3380, 3382, 3384, 3386, 3388, 3390,3392, 3394, 3396, 3398, 3400, 3402, 3404, 3406, 3408, 3410, 3412, 3414,3416, 3418, 3420, 3422, 3424, 3426, 3428, 3430, 3432, 3434, 3436, 3438,3440, 3442, 3444, 3446, 3448, 3450, 3452, 3454, 3456, 3458, 3460, 3462,3464, 3466, 3468, 3470, 3472, 3474, 3476, 3478, 3480, 3482, 3484, 3486,3488, and 3490. In some embodiments, the polypeptide sequence of theengineered glycosyltransferase comprises a sequence at least 95%identical to any of SEQ ID NOS: 3182, 3184, 3186, 3188, 3190, 3192,3194, 3196, 3198, 3200, 3202, 3204, 3206, 3208, 3210, 3212, 3214, 3216,3218, 3220, 3222, 3224, 3226, 3228, 3230, 3232, 3234, 3236, 3238, 3240,3242, 3244, 3246, 3248, 3250, 3252, 3254, 3256, 3258, 3260, 3262, 3264,3266, 3268, 3270, 3272, 3274, 3276, 3278, 3280, 3282, 3284, 3286, 3288,3290, 3292, 3294, 3296, 3298, 3300, 3302, 3304, 3306, 3308, 3310, 3312,3314, 3316, 3318, 3320, 3322, 3324, 3326, 3328, 3330, 3332, 3334, 3336,3338, 3340, 3342, 3344, 3346, 3348, 3350, 3352, 3354, 3356, 3358, 3360,3362, 3364, 3366, 3368, 3370, 3372, 3374, 3376, 3378, 3380, 3382, 3384,3386, 3388, 3390, 3392, 3394, 3396, 3398, 3400, 3402, 3404, 3406, 3408,3410, 3412, 3414, 3416, 3418, 3420, 3422, 3424, 3426, 3428, 3430, 3432,3434, 3436, 3438, 3440, 3442, 3444, 3446, 3448, 3450, 3452, 3454, 3456,3458, 3460, 3462, 3464, 3466, 3468, 3470, 3472, 3474, 3476, 3478, 3480,3482, 3484, 3486, 3488, and 3490. In some embodiments, the polypeptidesequence of the engineered glycosyltransferase comprises any of SEQ IDNOS: 3182, 3184, 3186, 3188, 3190, 3192, 3194, 3196, 3198, 3200, 3202,3204, 3206, 3208, 3210, 3212, 3214, 3216, 3218, 3220, 3222, 3224, 3226,3228, 3230, 3232, 3234, 3236, 3238, 3240, 3242, 3244, 3246, 3248, 3250,3252, 3254, 3256, 3258, 3260, 3262, 3264, 3266, 3268, 3270, 3272, 3274,3276, 3278, 3280, 3282, 3284, 3286, 3288, 3290, 3292, 3294, 3296, 3298,3300, 3302, 3304, 3306, 3308, 3310, 3312, 3314, 3316, 3318, 3320, 3322,3324, 3326, 3328, 3330, 3332, 3334, 3336, 3338, 3340, 3342, 3344, 3346,3348, 3350, 3352, 3354, 3356, 3358, 3360, 3362, 3364, 3366, 3368, 3370,3372, 3374, 3376, 3378, 3380, 3382, 3384, 3386, 3388, 3390, 3392, 3394,3396, 3398, 3400, 3402, 3404, 3406, 3408, 3410, 3412, 3414, 3416, 3418,3420, 3422, 3424, 3426, 3428, 3430, 3432, 3434, 3436, 3438, 3440, 3442,3444, 3446, 3448, 3450, 3452, 3454, 3456, 3458, 3460, 3462, 3464, 3466,3468, 3470, 3472, 3474, 3476, 3478, 3480, 3482, 3484, 3486, 3488, and3490.

The present invention also provides engineered glycosyltransferases,wherein the polypeptide sequence of the engineered glycosyltransferasescomprise at least one mutation or mutation set at one or more positionsselected from 106/164/172/200/409, 106/164/172/210/240/415,106/164/172/242/409/416, 106/164/200/210/242, 106/164/200/210/415,106/164/200/240/408/409/416, 106/164/210/240/408/409/416,106/164/210/408/409, 106/164/240/415, 106/164/409/415/423,106/172/200/210/240/242/408/415/416/423, 106/172/200/210/242,106/172/240, 106/172/240/242/409/415, 106/172/242, 106/172/242/416/423,106/172/408/409, 106/172/409/423, 106/200/210/409, 106/210/240,106/210/240/408/415, 106/240/242, 106/240/242/409, 106/242/408/409,164/172/200/210/242/409, 164/172/240/242/415, 164/172/242/408,164/200/408/415/416, 164/240/242/408, 164/240/242/415/423, 164/423,172/200/210/240/408, 172/200/240, 172/200/408/416, 172/210/415, 172/240,172/240/242/415, 172/240/409, 200/210/240/242/409, 200/240/242/407,200/242/415/416/423, 200/408/409, 210/240, 240, 240/242,240/242/408/416, 240/242/415/423, 240/408, 240/415, and 409/415, whereinthe positions are numbered with reference to SEQ ID NO: 3244. In someembodiments, the polypeptide sequence of the engineeredglycosyltransferase comprises at least one mutation or mutation set atone or more positions selected from 106A/164H/172R/200A/409L,106A/164H/172R/210V/240E/415R, 106A/164H/172R/242I/409L/416R,106A/164H/200A/210V/242I, 106A/164H/200A/210V/415A,106A/164H/200A/240E/408P/409L/416R, 106A/164H/210V/240E/408P/409L/416R,106A/164H/210V/408P/409L, 106A/164H/240E/415A, 106A/164H/409L/415A/423R,106A/172R/200A/210V/240E/242I/408P/415A/416R/423R,106A/172R/200A/210V/242I, 106A/172R/240E, 106A/172R/240E/242I/409L/415R,106A/172R/242I, 106A/172R/242I/416R/423R, 106A/172R/408P/409L,106A/172R/409L/423R, 106A/200A/210V/409L, 106A/210V/240E,106A/210V/240E/408P/415R, 106A/240E/242I, 106A/240E/242I/409L,106A/242I/408P/409L, 164H/172R/200A/210V/242I/409L,164H/172R/240E/242I/415R, 164H/172R/242I/408P, 164H/200A/408P/415A/416R,164H/240E/242I/408P, 164H/240E/242I/415R/423R, 164H/423R,172R/200A/210V/240E/408P, 172R/200A/240E, 172R/200A/408P/416R,172R/210V/415A, 172R/240E, 172R/240E/242I/415R, 172R/240E/409L,200A/210V/240E/242I/409L, 200A/240E/242I/407S, 200A/242I/415A/416R/423R,200A/408P/409L, 210V/240E, 240E, 240E/242I, 240E/242I/408P/416R,240E/242I/415A/423R, 240E/408P, 240E/415R, and 409L/415R, wherein thepositions are numbered with reference to SEQ ID NO: 3244. In someembodiments, the polypeptide sequence of the engineeredglycosyltransferase comprises at least one mutation or mutation set atone or more positions selected from K106A/V164H/N172R/G200A/T409L,K106A/V164H/N172R/E210V/N240E/G415R,K106A/V164H/N172R/A242I/T409L/K416R, K106A/V164H/G200A/E210V/A242I,K106A/V164H/G200A/E210V/G415A,K106A/V164H/G200A/N240E/E408P/T409L/K416R,K106A/V164H/E210V/N240E/E408P/T409L/K416R,K106A/V164H/E210V/E408P/T409L, K106A/V164H/N240E/G415A,K106A/V164H/T409L/G415A/N423R,K106A/N172R/G200A/E210V/N240E/A242I/E408P/G415A/K416R/N423R,K106A/N172R/G200A/E210V/A242I, K106A/N172R/N240E,K106A/N172R/N240E/A242I/T409L/G415R, K106A/N172R/A242I,K106A/N172R/A242I/K416R/N423R, K106A/N172R/E408P/T409L,K106A/N172R/T409L/N423R, K106A/G200A/E210V/T409L, K106A/E210V/N240E,K106A/E210V/N240E/E408P/G415R, K106A/N240E/A242I,K106A/N240E/A242I/T409L, K106A/A242I/E408P/T409L,V164H/N172R/G200A/E210V/A242I/T409L, V164H/N172R/N240E/A242I/G415R,V164H/N172R/A242I/E408P, V164H/G200A/E408P/G415A/K416R,V164H/N240E/A242I/E408P, V164H/N240E/A242I/G415R/N423R, V164H/N423R,N172R/G200A/E210V/N240E/E408P, N172R/G200A/N240E,N172R/G200A/E408P/K416R, N172R/E210V/G415A, N172R/N240E,N172R/N240E/A242I/G415R, N172R/N240E/T409L,G200A/E210V/N240E/A242I/T409L, G200A/N240E/A242I/G407S,G200A/A242I/G415A/K416R/N423R, G200A/E408P/T409L, E210V/N240E, N240E,N240E/A242I, N240E/A242I/E408P/K416R, N240E/A242I/G415A/N423R,N240E/E408P, N240E/G415R, and T409L/G415R, wherein the positions arenumbered with reference to SEQ ID NO: 3244. In some embodiments, thepolypeptide sequence of the engineered glycosyltransferase comprises atleast one mutation or mutation set at one or more positions selectedfrom 14, 35, 42, 46, 49, 105, 134, 143, 179, 181, 232, 278, 290, 336,373, 381, 401, and 441, wherein the positions are numbered withreference to SEQ ID NO: 3244. In some embodiments, the polypeptidesequence of the engineered glycosyltransferase comprises at least onemutation or mutation set at one or more positions selected from 14K,35D, 42F, 42I, 42V, 46T, 46V, 49A, 49M, 49P, 49Q, 49S, 105A, 134A, 134C,134S, 143P, 179A, 179D, 179T, 181L, 232T, 278I, 278L, 290L, 336A, 373R,381G, 401V, 4411, and 441R, wherein the positions are numbered withreference to SEQ ID NO: 3244. In some embodiments, the polypeptidesequence of the engineered glycosyltransferase comprises at least onemutation or mutation set at one or more positions selected from R14K,Q35D, L42F, L42I, L42V, C46T, C46V, L49A, L49M, L49P, L49Q, L49S, S105A,V134A, V134C, V134S, K143P, V179A, V179D, V179T, M181L, P232T, V278I,V278L, I290 μL, S336A, K373R, A381G, L401V, Q441I, and Q441R, whereinthe positions are numbered with reference to SEQ ID NO: 3244.

In some embodiments, the polypeptide sequence of the engineeredglycosyltransferase comprises a sequence at least 90% identical to anyof SEQ ID NOS: 3492, 3494, 3496, 3498, 3500, 3502, 3504, 3506, 3508,3510, 3512, 3514, 3516, 3518, 3520, 3522, 3524, 3526, 3528, 3530, 3532,3534, 3536, 3538, 3540, 3542, 3544, 3546, 3548, 3550, 3552, 3554, 3556,3558, 3560, 3562, 3564, 3566, 3568, 3570, 3572, 3574, 3576, 3578, 3580,3582, 3584, 3586, 3588, 3590, 3592, 3594, 3596, 3598, 3600, 3602, 3604,3606, 3608, 3610, 3612, 3614, 3616, 3618, 3620, 3622, 3624, 3626, 3628,3630, 3632, 3634, 3636, 3638, 3640, 3642, 3644, 3646, 3648, 3650, and3652. In some embodiments, the polypeptide sequence of the engineeredglycosyltransferase comprises a sequence at least 95% identical to anyof SEQ ID NOS: 3492, 3494, 3496, 3498, 3500, 3502, 3504, 3506, 3508,3510, 3512, 3514, 3516, 3518, 3520, 3522, 3524, 3526, 3528, 3530, 3532,3534, 3536, 3538, 3540, 3542, 3544, 3546, 3548, 3550, 3552, 3554, 3556,3558, 3560, 3562, 3564, 3566, 3568, 3570, 3572, 3574, 3576, 3578, 3580,3582, 3584, 3586, 3588, 3590, 3592, 3594, 3596, 3598, 3600, 3602, 3604,3606, 3608, 3610, 3612, 3614, 3616, 3618, 3620, 3622, 3624, 3626, 3628,3630, 3632, 3634, 3636, 3638, 3640, 3642, 3644, 3646, 3648, 3650, and3652. In some embodiments, the polypeptide sequence of the engineeredglycosyltransferase comprises any of SEQ ID NOS: 3492, 3494, 3496, 3498,3500, 3502, 3504, 3506, 3508, 3510, 3512, 3514, 3516, 3518, 3520, 3522,3524, 3526, 3528, 3530, 3532, 3534, 3536, 3538, 3540, 3542, 3544, 3546,3548, 3550, 3552, 3554, 3556, 3558, 3560, 3562, 3564, 3566, 3568, 3570,3572, 3574, 3576, 3578, 3580, 3582, 3584, 3586, 3588, 3590, 3592, 3594,3596, 3598, 3600, 3602, 3604, 3606, 3608, 3610, 3612, 3614, 3616, 3618,3620, 3622, 3624, 3626, 3628, 3630, 3632, 3634, 3636, 3638, 3640, 3642,3644, 3646, 3648, 3650, and 3652.

The present invention also provides engineered glycosyltransferases,wherein the polypeptide sequence of the engineered glycosyltransferasescomprise at least one mutation or mutation set at one or more positionsselected from 21, 91, 125, 127, 130/187, 143, 143/150, 145, 152, 156,186, 187, 195, 197, 200, 201, 202, 264, 268, 364, 365, and 415, whereinthe positions are numbered with reference to SEQ ID NO: 3346. In someembodiments, the polypeptide sequence of the engineeredglycosyltransferase comprises at least one mutation or mutation set atone or more positions selected from 21M, 21P, 91M, 125C, 125M, 125V,127G, 130T/187Q, 143H, 143P/150S, 145W, 152L, 156P, 156Q, 186N, 186V,187S, 195F, 195G, 195R, 195S, 195T, 195Y, 197D, 197L, 197Q, 197W, 200E,200P, 200R, 200T, 201A, 201R, 201S, 201W, 202A, 202W, 264S, 264T, 268F,268Q, 268W, 364S, 364W, 365L, and 415D, wherein the positions arenumbered with reference to SEQ ID NO: 3346. In some embodiments, thepolypeptide sequence of the engineered glycosyltransferase comprises atleast one mutation or mutation set at one or more positions selectedfrom L21M, L21P, P91M, F125C, F125M, F125V, Q127G, A130T/K187Q, K143H,K143P/A150S, L145W, F152L, F156P, F156Q, E186N, E186V, K187S, L195F,L195G, L195R, L195S, L195T, L195Y, P197D, P197L, P197Q, P197W, G200E,G200P, G200R, G200T, G201A, G201R, G201S, G201W, I202A, I202W, F264S,F264T, Y268F, Y268Q, Y268W, M364S, M364W, Q365L, and G415D, wherein thepositions are numbered with reference to SEQ ID NO: 3346. In someembodiments, the polypeptide sequence of the engineeredglycosyltransferase comprises a sequence at least 90% identical to anyof SEQ ID NOS: 3760, 3762, 3764, 3766, 3768, 3770, 3772, 3774, 3776,3778, 3780, 3782, 3784, 3786, 3788, 3790, 3792, 3794, 3796, 3798, 3800,3802, 3804, 3806, 3808, 3810, 3812, 3814, 3816, 3818, 3820, 3822, 3824,3826, 3828, 3830, 3832, 3834, 3836, 3838, 3840, 3842, 3844, 3846, 3848,and 3850. In some embodiments, the polypeptide sequence of theengineered glycosyltransferase comprises a sequence at least 95%identical to any of SEQ ID NOS: 3760, 3762, 3764, 3766, 3768, 3770,3772, 3774, 3776, 3778, 3780, 3782, 3784, 3786, 3788, 3790, 3792, 3794,3796, 3798, 3800, 3802, 3804, 3806, 3808, 3810, 3812, 3814, 3816, 3818,3820, 3822, 3824, 3826, 3828, 3830, 3832, 3834, 3836, 3838, 3840, 3842,3844, 3846, 3848, and 3850. In some embodiments, the polypeptidesequence of the engineered glycosyltransferase any of SEQ ID NOS: 3760,3762, 3764, 3766, 3768, 3770, 3772, 3774, 3776, 3778, 3780, 3782, 3784,3786, 3788, 3790, 3792, 3794, 3796, 3798, 3800, 3802, 3804, 3806, 3808,3810, 3812, 3814, 3816, 3818, 3820, 3822, 3824, 3826, 3828, 3830, 3832,3834, 3836, 3838, 3840, 3842, 3844, 3846, 3848, and 3850.

The present invention also provides engineered glycosyltransferases,wherein the polypeptide sequence of the engineered glycosyltransferasescomprise at least one mutation or mutation set at one or more positionsselected from 2/5/113/186/237/406, 2/73/186/237, 2/189/286,2/286/355/416/422, 5/34/73/113/406, 5/73/186/406, 5/113/237/406,5/186/237/245/256/406, 5/186/237/406, 5/256/406, 34/113/186/237/406,72/73/172/235/240/242/438, 72/172/239/240/242/408, 72/172/240/242,73/172, 73/172/235/239/240, 73/172/235/239/242, 73/172/239/240,73/172/239/240/242, 73/172/240/242/408, 73/172/240/408, 73/186/237/406,73/235/240/323, 73/235/240/408, 73/237, 73/239/242, 113/186/406, 172,172/188/323, 172/235, 172/235/239/240/242, 172/235/239/408, 172/235/240,172/235/240/242/438, 172/239/240/242/323/408, 172/240, 172/240/299/323,186, 186/237, 186/237/286, 186/406, 189/333/355/421, 235/239/240,235/240/242, 237, 239/240, 239/240/242/256/323, 239/240/242/323/408,239/240/408, 239/242/408, 240/242/256/438, and 242/408, wherein thepositions are numbered with reference to SEQ ID NO: 3502. In someembodiments, the polypeptide sequence of the engineeredglycosyltransferase comprises at least one mutation or mutation set atone or more positions selected from 2-/5N/113I/186G/237V/406Q,2-/73A/186G/237V, 2S/189S/286Q, 2S/286Q/355A/416R/422R,5N/34R/73A/113I/406N, 5N/73A/186G/406N, 5N/113I/237V/406N,5N/186G/237V/245T/256I/406Q, 5N/186G/237V/406Q, 5N/256L/406Q,34R/113I/186G/237L/406N, 72Y/73P/172R/235M/240E/242I/438T,72Y/172R/239A/240E/242I/408P, 72Y/172R/240E/242I, 73A/186G/237L/406Q,73A/237V, 73P/172R, 73P/172R/235M/239A/240E, 73P/172R/235M/239A/242I,73P/172R/239A/240E, 73P/172R/239A/240E/242I, 73P/172R/240E/242I/408P,73P/172R/240E/408P, 73P/235M/240E/323P, 73P/235M/240E/408P,73P/239A/242I, 113I/186G/406Q, 172R, 172R/188G/323P, 172R/235M,172R/235M/239A/240E/242I, 172R/235M/239A/408P, 172R/235M/240E,172R/235M/240E/242I/438T, 172R/239A/240E/242I/323P/408P, 172R/240E,172R/240E/299A/323P, 186G, 186G/237L, 186G/237V/286S, 186G/406N,186G/406Q, 189S/333H/355A/421Q, 235M/239A/240E, 235M/240E/242I, 237L,239A/240E, 239A/240E/242I/256S/323P, 239A/240E/242I/323P/408P,239A/240E/408P, 239A/242I/408P, 240E/242I/256S/438T, and 242I/408P,wherein the positions are numbered with reference to SEQ ID NO: 3502. Insome embodiments, the polypeptide sequence of the engineeredglycosyltransferase comprises at least one mutation or mutation set atone or more positions selected from H2-/H5N/L113I/E186G/P237V/T406Q,H2-/H73A/E186G/P237V, H2S/P189S/N286Q, H2S/N286Q/F355A/K416R/K422R,H5N/K34R/H73A/L113I/T406N, H5N/H73A/E186G/T406N, H5N/L113I/P237V/T406N,H5N/E186G/P237V/K245T/E256I/T406Q, H5N/E186G/P237V/T406Q,H5N/E256L/T406Q, K34R/L113I/E186G/P237L/T406N,L72Y/H73P/N172R/Q235M/N240E/A242I/E438T,L72Y/N172R/T239A/N240E/A242I/E408P, L72Y/N172R/N240E/A242I,H73A/E186G/P237L/T406Q, H73A/P237V, H73P/N172R,H73P/N172R/Q235M/T239A/N240E, H73P/N172R/Q235M/T239A/A242I,H73P/N172R/T239A/N240E, H73P/N172R/T239A/N240E/A242I,H73P/N172R/N240E/A242I/E408P, H73P/N172R/N240E/E408P,H73P/Q235M/N240E/D323P, H73P/Q235M/N240E/E408P, H73P/T239A/A242I,L113I/E186G/T406Q, N172R, N172R/E188G/D323P, N172R/Q235M,N172R/Q235M/T239A/N240E/A242I, N172R/Q235M/T239A/E408P,N172R/Q235M/N240E, N172R/Q235M/N240E/A242I/E438T,N172R/T239A/N240E/A242I/D323P/E408P, N172R/N240E,N172R/N240E/E299A/D323P, E186G, E186G/P237L, E186G/P237V/N286S,E186G/T406N, E186G/T406Q, P189S/N333H/F355A/S421Q, Q235M/T239A/N240E,Q235M/N240E/A242I, P237L, T239A/N240E, T239A/N240E/A242I/E256S/D323P,T239A/N240E/A242I/D323P/E408P, T239A/N240E/E408P, T239A/A242I/E408P,N240E/A242I/E256S/E438T, and A242I/E408P, wherein the positions arenumbered with reference to SEQ ID NO: 3502. In some embodiments, thepolypeptide sequence of the engineered glycosyltransferase comprises atleast one mutation or mutation set at one or more positions selectedfrom 96, 127, 132, 144, 153, 155, 156, 186, 187, 196, 199, and 200,wherein the positions are numbered with reference to SEQ ID NO: 3502. Insome embodiments, the polypeptide sequence of the engineeredglycosyltransferase comprises at least one mutation or mutation set atone or more positions selected from 96A, 96P, 1271, 127L, 127V, 132H,132K, 132T, 144V, 153A, 153G, 155M, 156W, 186A, 186G, 186R, 187A, 187R,187T, 196S, 199A, 199S, 199Y, and 200S, wherein the positions arenumbered with reference to SEQ ID NO: 3502. In some embodiments, thepolypeptide sequence of the engineered glycosyltransferase comprises atleast one mutation or mutation set at one or more positions selectedfrom G96A, G96P, Q127I, Q127L, Q127V, Q132H, Q132K, Q132T, I144V, S153A,S153G, F155M, F156W, E186A, E186G, E186R, K187A, K187R, K187T, A196S,N199A, N199S, N199Y, and A200S, wherein the positions are numbered withreference to SEQ ID NO: 3502.

In some embodiments, the polypeptide sequence of the engineeredglycosyltransferase comprises a sequence at least 90% identical to anyof SEQ ID NOS: 3654, 3656, 3658, 3660, 3662, 3664, 3666, 3668, 3670,3672, 3674, 3676, 3678, 3680, 3682, 3684, 3686, 3688, 3690, 3692, 3694,3696, 3698, 3700, 3702, 3704, 3706, 3708, 3710, 3712, 3714, 3716, 3718,3720, 3722, 3724, 3726, 3728, 3730, 3732, 3734, 3736, 3738, 3740, 3742,3744, 3746, 3748, 3750, 3752, 3754, 3756, 3758, 3852, 3854, 3856, 3858,3860, 3862, 3864, 3866, 3868, 3870, 3872, 3874, 3876, 3878, 3880, 3882,3884, 3886, 3888, 3890, 3892, 3894, 3896, and 3898. In some embodiments,the polypeptide sequence of the engineered glycosyltransferase comprisesa sequence at least 95% identical to any of SEQ ID NOS: 3654, 3656,3658, 3660, 3662, 3664, 3666, 3668, 3670, 3672, 3674, 3676, 3678, 3680,3682, 3684, 3686, 3688, 3690, 3692, 3694, 3696, 3698, 3700, 3702, 3704,3706, 3708, 3710, 3712, 3714, 3716, 3718, 3720, 3722, 3724, 3726, 3728,3730, 3732, 3734, 3736, 3738, 3740, 3742, 3744, 3746, 3748, 3750, 3752,3754, 3756, 3758, 3852, 3854, 3856, 3858, 3860, 3862, 3864, 3866, 3868,3870, 3872, 3874, 3876, 3878, 3880, 3882, 3884, 3886, 3888, 3890, 3892,3894, 3896, and 3898. In some embodiments, the polypeptide sequence ofthe engineered glycosyltransferase comprises any of SEQ ID NOS: 3654,3656, 3658, 3660, 3662, 3664, 3666, 3668, 3670, 3672, 3674, 3676, 3678,3680, 3682, 3684, 3686, 3688, 3690, 3692, 3694, 3696, 3698, 3700, 3702,3704, 3706, 3708, 3710, 3712, 3714, 3716, 3718, 3720, 3722, 3724, 3726,3728, 3730, 3732, 3734, 3736, 3738, 3740, 3742, 3744, 3746, 3748, 3750,3752, 3754, 3756, 3758, 3852, 3854, 3856, 3858, 3860, 3862, 3864, 3866,3868, 3870, 3872, 3874, 3876, 3878, 3880, 3882, 3884, 3886, 3888, 3890,3892, 3894, 3896, and 3898.

The present invention also provides engineered glycosyltransferases,wherein the polypeptide sequence of the engineered glycosyltransferasescomprise at least one mutation or mutation set at one or more positionsselected from 2/5/35/105/143/237/373/416/422,2/5/35/143/232/237/416/422/441, 2/5/35/232/278/373/416,2/5/105/143/232/373/416/422, 2/5/278, 2/105/143/232/237/278/373/441,2/143/232/373/441, 2/143/373/441, 5/35/232/373/416/422,5/105/232/237/373/416/441, 5/105/237/278/422/441, 5/105/237/416/422/441,5/143/189/232/237/441, 5/143/232/237/278, 5/143/232/237/416/422,5/143/232/373/422/441, 5/143/237/278/373/416/422, 5/143/373,5/189/237/278/373/416/422/441, 5/232/416/422, 5/237/373/422, 5/373/416,26/42, 26/42/46/49, 26/42/46/49/134, 26/42/46/49/134/186, 26/42/46/134,26/42/49/134, 26/42/49/134/186/355, 26/42/49/134/401, 26/42/134/401,26/49/134, 26/134, 26/134/186/355/401, 26/134/355, 26/134/401,26/355/401, 35, 35/105/189/232/373/416, 42/46/49/97/134/401,42/46/49/134, 42/46/49/134/186, 42/46/49/134/186/355,42/46/49/134/235/355/401, 42/46/49/134/401, 42/46/49/186,42/49/134/186/355, 42/49/134/186/401, 42/49/134/355/401, 42/134/186,42/186/355, 46/49/134, 49/134/355, 105/143/232/237/373/416/422/441,105/143/278/373, 105/189/237, 105/237/278/373/416/422,105/237/373/422/441, 105/373, 105/373/441, 134/401, 143, 143/189,143/189/232/422/441, 143/189/237/373/416/422, 143/232, 143/237,143/237/422/441, 186/355, 189/232/373/416/422/441, 189/237/278/416/441,232/237, 237/373, 237/373/441, and 237/422, wherein the positions arenumbered with reference to SEQ ID NO: 3696. In some embodiments, thepolypeptide sequence of the engineered glycosyltransferase comprises atleast one mutation or mutation set at one or more positions selectedfrom 2-/5N/35D/105A/143P/237V/373R/416R/422R,2-/5N/35D/143P/232T/237L/416R/422R/441R, 2-/5N/35D/232T/278L/373R/416R,2-/5N/105A/143P/232T/373R/416R/422R, 2-/5N/278L,2-/105A/143P/232T/237L/278L/373R/441R, 2-/143P/232T/373R/441R,2-/143P/373R/441R, 5N/35D/232T/373R/416R/422R,5N/105A/232T/237L/373R/416R/441R, 5N/105A/237L/278I/422R/441R,5N/105A/237V/416R/422R/441R, 5N/143P/189S/232T/237V/441R,5N/143P/232T/237L/278L, 5N/143P/232T/237L/416R/422R,5N/143P/232T/373R/422R/441R, 5N/143P/237L/278L/373R/416R/422R,5N/143P/373R, 5N/189S/237V/278I/373R/416R/422R/441R, 5N/232T/416R/422R,5N/237L/373R/422R, 5N/373R/416R, 26V/42I/49Q/134C/186G/355A,26V/42I/49Q/134C/401V, 26V/42V, 26V/42V/46V/49A, 26V/42V/46V/49A/134A,26V/42V/46V/49A/134C/186G, 26V/42V/46V/134A, 26V/42V/49S/134C,26V/42V/134C/401V, 26V/49Q/134A, 26V/134A, 26V/134A/186G/355A/401V,26V/134C/355A, 26V/134T/401V, 26V/355A/401V, 35D,35D/105A/189S/232T/373R/416R, 42I/46V/49A/134A, 42I/46V/49S/134C/186G,42I/46V/49S/186G, 42V/46V/49A/134A/401V, 42V/46V/49A/134T/186G/355A,42V/46V/49P/134C/235R/355A/401V, 42V/46V/49S/97P/134C/401V,42V/49A/134C/186G/355A, 42V/49S/134A/186G/401V, 42V/49S/134C/355A/401V,42V/134C/186G, 42V/186G/355A, 46V/49Q/134T, 49S/134C/355A,105A/143P/232T/237V/373R/416R/422R/441R, 105A/143P/278L/373R,105A/189S/237V, 105A/237L/278L/373R/416R/422R, 105A/237L/373R/422R/441R,105A/373R, 105A/373R/441R, 134C/401V, 143P, 143P/1895,143P/189/232T/422R/441R, 143P/189/237L/373R/416R/422R, 143P/232T,143P/237L/422R/441R, 143P/237V, 186G/355A, 189/232T/373R/416R/422R/441R,1895/237L/278I/416R/441R, 232T/237L, 237L/422R, 237V/373R, and237V/373R/441R, wherein the positions are numbered with reference to SEQID NO: 3696. In some embodiments, the polypeptide sequence of theengineered glycosyltransferase comprises at least one mutation ormutation set at one or more positions selected fromH2-/H5N/Q35D/S105A/K143P/P237V/K373R/K416R/K422R,H2-/H5N/Q35D/K143P/P232T/P237L/K416R/K422R/Q441R,H2-/H5N/Q35D/P232T/V278L/K373R/K416R,H2-/H5N/S105A/K143P/P232T/K373R/K416R/K422R, H2-/H5N/V278L,H2-/S105A/K143P/P232T/P237L/V278L/K373R/Q441R,H2-/K143P/P232T/K373R/Q441R, H2-/K143P/K373R/Q441R,H5N/Q35D/P232T/K373R/K416R/K422R,H5N/S105A/P232T/P237L/K373R/K416R/Q441R,H5N/S105A/P237L/V278I/K422R/Q441R, H5N/S105A/P237V/K416R/K422R/Q441R,H5N/K143P/P189S/P232T/P237V/Q441R, H5N/K143P/P232T/P237L/V278L,H5N/K143P/P232T/P237L/K416R/K422R, H5N/K143P/P232T/K373R/K422R/Q441R,H5N/K143P/P237L/V278L/K373R/K416R/K422R, H5N/K143P/K373R,H5N/P189S/P237V/V278I/K373R/K416R/K422R/Q441R, H5N/P232T/K416R/K422R,H5N/P237L/K373R/K422R, H5N/K373R/K416R,I26V/L42I/L49Q/V134C/E186G/F355A, I26V/L42I/L49Q/V134C/L401V, I26V/L42V,I26V/L42V/C46V/L49A, I26V/L42V/C46V/L49A/V134A,I26V/L42V/C46V/L49A/V134C/E186G, I26V/L42V/C46V/V134A,I26V/L42V/L49S/V134C, I26V/L42V/V134C/L401V, I26V/L49Q/V134A,I26V/V134A, I26V/V134A/E186G/F355A/L401V, I26V/V134C/F355A,I26V/V134T/L401V, I26V/F355A/L401V, Q35D,Q35D/S105A/P189S/P232T/K373R/K416R, L42I/C46V/L49A/V134A,L42I/C46V/L49S/V134C/E186G, L42I/C46V/L49S/E186G,L42V/C46V/L49A/V134A/L401V, L42V/C46V/L49A/V134T/E186G/F355A,L42V/C46V/L49P/V134C/Q235R/F355A/L401V, L42V/C46V/L49S/T97P/V134C/L401V,L42V/L49A/V134C/E186G/F355A, L42V/L49S/V134A/E186G/L401V,L42V/L49S/V134C/F355A/L401V, L42V/V134C/E186G, L42V/E186G/F355A,C46V/L49Q/V134T, L49S/V134C/F355A,S105A/K143P/P232T/P237V/K373R/K416R/K422R/Q441R,S105A/K143P/V278L/K373R, S105A/P189S/P237V,S105A/P237L/V278L/K373R/K416R/K422R, S105A/P237L/K373R/K422R/Q441R,S105A/K373R, S105A/K373R/Q441R, V134C/L401V, K143P, K143P/P189S,K143P/P189S/P232T/K422R/Q441R, K143P/P189S/P237L/K373R/K416R/K422R,K143P/P232T, K143P/P237L/K422R/Q441R, K143P/P237V, E186G/F355A,P189S/P232T/K373R/K416R/K422R/Q441R, P189S/P237L/V278I/K416R/Q441R,P232T/P237L, P237L/K422R, P237V/K373R, and P237V/K373R/Q441R, whereinthe positions are numbered with reference to SEQ ID NO: 3696. In someembodiments, the polypeptide sequence of the engineeredglycosyltransferase comprises at least one mutation or mutation set atone or more positions selected from 3, 8, 50, 61, 62, 101, 137, 158,161, 164, 176, 193, 223, 223/243, 235, 237, 239, 240, 243, 244, 248,249, 301, 323, 330, 352, 364, 426, and 427, wherein the positions arenumbered with reference to SEQ ID NO: 3696. In some embodiments, thepolypeptide sequence of the engineered glycosyltransferase comprises atleast one mutation or mutation set at one or more positions selectedfrom 3N, 8R, 8V, 50P, 50V, 61N, 61Y, 62A, 101L, 137R, 158W, 161L, 164A,164E, 164F, 164L, 176P, 176Q, 176T, 193F, 223A, 223L/243G, 235R, 237A,237L, 239F, 239G, 239M, 239P, 239Q, 239Y, 240V, 243G, 243H, 243R, 243S,243Y, 244N, 248T, 249G, 249H, 301A, 323T, 330C, 352A, 364Q, 426L, 427S,and 427T, wherein the positions are numbered with reference to SEQ IDNO: 3696. In some embodiments, the polypeptide sequence of theengineered glycosyltransferase comprises at least one mutation ormutation set at one or more positions selected from H3N, S8R, S8V, I50P,I50V, E61N, E61Y, K62A, A101L, Q137R, F158W, R161L, H164A, H164E, H164F,H164L, R176P, R176Q, R176T, D193F, T223A, T223L/D243G, Q235R, P237A,P237L, T239F, T239G, T239M, T239P, T239Q, T239Y, E240V, D243G, D243H,D243R, D243S, D243Y, D244N, I248T, D249G, D249H, Q301A, D323T, H330C,S352A, M364Q, A426L, R427S, and R427T, wherein the positions arenumbered with reference to SEQ ID NO: 3696. In some embodiments, thepolypeptide sequence of the engineered glycosyltransferase comprises asequence at least 90% identical to any of SEQ ID NOS: 3900, 3902, 3904,3906, 3908, 3910, 3912, 3914, 3916, 3918, 3920, 3922, 3924, 3926, 3928,3930, 3932, 3934, 3936, 3938, 3940, 3942, 3944, 3946, 3948, 3950, 3952,3954, 3956, 3958, 3960, 3962, 3964, 3966, 3968, 3970, 3972, 3974, 3976,3978, 3980, 3982, 3984, 3986, 3988, 3990, 3992, 3994, 3996, 3998, 4000,4002, 4004, 4006, 4008, 4010, 4012, 4014, 4016, 4018, 4020, 4022, 4024,4026, 4028, 4030, 4032, 4034, 4036, 4038, 4040, 4042, 4044, 4046, 4048,4050, 4052, 4054, 4056, 4058, 4060, 4062, 4064, 4066, 4068, 4070, 4072,4074, 4076, 4078, 4080, 4082, 4084, 4086, 4088, 4090, 4092, 4094, 4096,4098, 4100, 4102, 4104, 4106, 4108, 4110, 4112, 4114, 4116, 4118, 4120,4122, 4124, 4126, 4128, 4130, 4132, 4134, 4136, 4138, 4140, 4142, 4144,and 4146. In some embodiments, the polypeptide sequence of theengineered glycosyltransferase comprises a sequence at least 95%identical to any of SEQ ID NOS: 3900, 3902, 3904, 3906, 3908, 3910,3912, 3914, 3916, 3918, 3920, 3922, 3924, 3926, 3928, 3930, 3932, 3934,3936, 3938, 3940, 3942, 3944, 3946, 3948, 3950, 3952, 3954, 3956, 3958,3960, 3962, 3964, 3966, 3968, 3970, 3972, 3974, 3976, 3978, 3980, 3982,3984, 3986, 3988, 3990, 3992, 3994, 3996, 3998, 4000, 4002, 4004, 4006,4008, 4010, 4012, 4014, 4016, 4018, 4020, 4022, 4024, 4026, 4028, 4030,4032, 4034, 4036, 4038, 4040, 4042, 4044, 4046, 4048, 4050, 4052, 4054,4056, 4058, 4060, 4062, 4064, 4066, 4068, 4070, 4072, 4074, 4076, 4078,4080, 4082, 4084, 4086, 4088, 4090, 4092, 4094, 4096, 4098, 4100, 4102,4104, 4106, 4108, 4110, 4112, 4114, 4116, 4118, 4120, 4122, 4124, 4126,4128, 4130, 4132, 4134, 4136, 4138, 4140, 4142, 4144, and 4146. In someembodiments, the polypeptide sequence of the engineeredglycosyltransferase comprises any of SEQ ID NOS: 3900, 3902, 3904, 3906,3908, 3910, 3912, 3914, 3916, 3918, 3920, 3922, 3924, 3926, 3928, 3930,3932, 3934, 3936, 3938, 3940, 3942, 3944, 3946, 3948, 3950, 3952, 3954,3956, 3958, 3960, 3962, 3964, 3966, 3968, 3970, 3972, 3974, 3976, 3978,3980, 3982, 3984, 3986, 3988, 3990, 3992, 3994, 3996, 3998, 4000, 4002,4004, 4006, 4008, 4010, 4012, 4014, 4016, 4018, 4020, 4022, 4024, 4026,4028, 4030, 4032, 4034, 4036, 4038, 4040, 4042, 4044, 4046, 4048, 4050,4052, 4054, 4056, 4058, 4060, 4062, 4064, 4066, 4068, 4070, 4072, 4074,4076, 4078, 4080, 4082, 4084, 4086, 4088, 4090, 4092, 4094, 4096, 4098,4100, 4102, 4104, 4106, 4108, 4110, 4112, 4114, 4116, 4118, 4120, 4122,4124, 4126, 4128, 4130, 4132, 4134, 4136, 4138, 4140, 4142, 4144, and4146.

The present invention also provides engineered glycosyltransferases,wherein the polypeptide sequence of the engineered glycosyltransferasescomprise at least one mutation or mutation set at one or more positionsselected from 5/73/113/186/187/373/423, 5/73/143/144/179/186/187/422,5/73/144/179/186/373/423, 5/73/144/187/373/422,5/143/144/179/181/186/187/373, 5/144/179/181/373/422, 5/144/373/422,49/96/127/132/156/196, 49/96/127/153/278,49/96/132/153/155/156/237/278/406, 49/96/132/153/199/200, 49/96/132/155,49/96/132/155/237/278, 49/96/155/199/200/406, 49/153/155,73/113/143/179/181/422, 73/113/179/181/186/187,73/143/144/179/186/187/373/423, 73/179/181/186/187/373,73/179/181/186/373/422, 73/181/186/187, 96/127/132/153/278,96/127/153/155, 96/132/153/155, 96/132/153/155/156,96/132/153/155/156/200, 96/132/153/156, 96/132/196/199, 96/132/278,96/153/155/156, 96/153/155/199/200/237, 96/153/406, 113/143/179/186/187,113/144/186/423, 113/144/373, 113/181/186/373/422, 113/373/422, 127/132,127/132/153/156, 127/132/155/156/406, 127/153/155/199/200/237, 127/406,132/153/155, 132/153/237/406, 132/155, 132/237,143/144/179/181/186/187/422, 143/179/181/186/187/422/423,144/179/181/186/187/373, 144/179/186/187/373, 153/155/156/237,153/155/196/199/237, 153/199/406, 153/237, 155/199, 179/181/186,179/181/186/187/423, 179/186/187, 179/187/373/422, 181/186/187/422/423,and 373/423, wherein the positions are numbered with reference to SEQ IDNO: 3956. In some embodiments, the polypeptide sequence of theengineered glycosyltransferase comprises at least one mutation ormutation set at one or more positions selected from5N/73A/113I/186G/187R/373R/423R, 5N/73A/143P/144V/179D/186G/187R/422R,5N/73A/144V/179D/186A/373R/423R, 5N/73A/144V/187R/373R/422R,5N/143P/144V/179T/181L/186G/187R/373R, 5N/144V/179T/181L/373R/422R,5N/144V/373R/422R, 49S/96A/127I/132K/156W/196S,49S/96A/132H/153A/199S/200S, 49S/96P/127I/153A/278L,49S/96P/132H/153A/155M/156W/237L/278L/406Q, 49S/96P/132H/155M,49S/96P/132K/155M/237L/278L, 49S/96P/155M/199S/200S/406Q, 49S/153A/155M,73A/113I/143P/179T/181L/422R, 73A/113I/179T/181L/186A/187R,73A/143P/144V/179D/186G/187T/373R/423R, 73A/179D/181L/186A/373R/422R,73A/179D/181L/186G/187R/373R, 73A/181L/186A/187T, 96A/132H/153A/156W,96A/132K/153A/155M/156W, 96A/132K/153A/155M/156W/200S,96A/153A/155M/156W, 96P/127I/132H/153A/278L, 96P/127I/153A/155M,96P/132H/153A/155M/156W, 96P/132H/196S/1995, 96P/132K/153A/155M,96P/132K/278L, 96P/153A/155M/199S/200S/237L, 96P/153A/406Q,113I/143P/179D/186G/187R, 113I/144V/186G/423R, 113I/144V/373R,113I/181L/186A/373R/422R, 113I/373R/422R, 127I/132H,127I/132H/153A/156W, 127I/132H/155M/156W/406Q,127I/153A/155M/199S/200S/237L, 127I/406Q, 132H/153A/237L/406Q,132H/155M, 132K/153A/155M, 132K/237L,143P/144V/179T/181L/186G/187R/422R, 143P/179T/181L/186G/187R/422R/423R,144V/179T/181L/186A/187R/373R, 144V/179T/186A/187T/373R,153A/155M/156W/237L, 153A/155M/196S/199S/237L, 153A/199S/406Q,153A/237L, 155M/199S, 179D/181L/186G/187R/423R, 179D/187T/373R/422R,179T/181L/186A, 179T/186A/187T, 181L/186G/187R/422R/423R, and 373R/423R,wherein the positions are numbered with reference to SEQ ID NO: 3956. Insome embodiments, the polypeptide sequence of the engineeredglycosyltransferase comprises at least one mutation or mutation set atone or more positions selected fromH5N/P73A/L113I/E186G/K187R/K373R/N423R,H5N/P73A/K143P/I144V/V179D/E186G/K187R/K422R,H5N/P73A/I144V/V179D/E186A/K373R/N423R,H5N/P73A/I144V/K187R/K373R/K422R,H5N/K143P/I144V/V179T/M181L/E186G/K187R/K373R,H5N/I144V/V179T/M181L/K373R/K422R, H5N/I144V/K373R/K422R,A49S/G96A/Q127I/Q132K/F156W/A196S, A49S/G96A/Q132H/S153A/N199S/A200S,A49S/G96P/Q127I/S153A/V278L,A49S/G96P/Q132H/S153A/F155M/F156W/P237L/V278L/T406Q,A49S/G96P/Q132H/F155M, A49S/G96P/Q132K/F155M/P237L/V278L,A49S/G96P/F155M/N199S/A200S/T406Q, A49S/S153A/F155M,P73A/L113I/K143P/V179T/M181L/K422R, P73A/L113I/V179T/M181L/E186A/K187R,P73A/K143P/I144V/V179D/E186G/K187T/K373R/N423R,P73A/V179D/M181L/E186A/K373R/K422R, P73A/V179D/M181L/E186G/K187R/K373R,P73A/M181L/E186A/K187T, G96A/Q132H/S153A/F156W,G96A/Q132K/S153A/F155M/F156W, G96A/Q132K/S153A/F155M/F156W/A200S,G96A/S153A/F155M/F156W, G96P/Q127I/Q132H/S153A/V278L,G96P/Q127I/S153A/F155M, G96P/Q132H/S153A/F155M/F156W,G96P/Q132H/A196S/N199S, G96P/Q132K/S153A/F155M, G96P/Q132K/V278L,G96P/S153A/F155M/N199S/A200S/P237L, G96P/S153A/T406Q,L113I/K143P/V179D/E186G/K187R, L113I/I144V/E186G/N423R,L113I/I144V/K373R, L113I/M181L/E186A/K373R/K422R, L113I/K373R/K422R,Q127I/Q132H, Q127I/Q132H/S153A/F156W, Q127I/Q132H/F155M/F156W/T406Q,Q127I/S153A/F155M/N199S/A200S/P237L, Q127I/T406Q,Q132H/S153A/P237L/T406Q, Q132H/F155M, Q132K/S153A/F155M, Q132K/P237L,K143P/I144V/V179T/M181L/E186G/K187R/K422R,K143P/V179T/M181L/E186G/K187R/K422R/N423R,I144V/V179T/M181L/E186A/K187R/K373R, I144V/V179T/E186A/K187T/K373R,S153A/F155M/F156W/P237L, S153A/F155M/A196S/N199S/P237L,S153A/N199S/T406Q, S153A/P237L, F155M/N199S,V179D/M181L/E186G/K187R/N423R, V179D/K187T/K373R/K422R,V179T/M181L/E186A, V179T/E186A/K187T, M181L/E186G/K187R/K422R/N423R, andK373R/N423R, wherein the positions are numbered with reference to SEQ IDNO: 3956. In some embodiments, the polypeptide sequence of theengineered glycosyltransferase comprises at least one mutation ormutation set at one or more positions selected from 11, 12, 41, 44,44/187, 45, 55, 56, 57, 65, 66, 70, 72, 73, 74/238, 82, 83, 85, 103,111, 113, 114, 117, 132, 135, 138, 140, 159, 160, 162, 167, 182, 214,220, 222, 223, 226, 236, 238, 256, 286, 299, 309, 387, 388, 389, 391,393, 406, 408, 412, 418, 422, 429, 430, 449, and 450, wherein thepositions are numbered with reference to SEQ ID NO: 3956. In someembodiments, the polypeptide sequence of the engineeredglycosyltransferase comprises at least one mutation or mutation set atone or more positions selected from 11D, 11L, 12, 12L, 41H, 44H,44S/187T, 45Y, 55A, 55G, 55L, 56R, 57G, 57H, 57P, 65C, 65G, 65P, 65Q,65R, 66L, 70W, 72V, 73H, 73S, 74M/238V, 82S, 83R, 85W, 103H, 103Q, IIF,113S, 114G, 114I, 117N, 117S, 132G, 135E, 138K, 1385, 138T, 140C, 140G,159N, 160D, 160Q, 160T, 162L, 167L, 1821, 182L, 182Q, 182T, 182V, 182Y,214H, 214T, 220G, 2205, 222F, 223D, 226Q, 236L, 236T, 236V, 238A, 238G,238I, 238R, 238S, 238T, 256R, 286G, 299V, 309P, 309R, 3095, 309T, 387P,387Q, 387R, 388A, 388S, 389A, 389G, 389L, 389V, 391A, 391L, 3915, 391V,393P, 393T, 406G, 4065, 408L, 408T, 412G, 412R, 418A, 418H, 418M, 422L,429D, 430P, 449G, 449H, 449L, 449R, 449S, 450N, and 450V, wherein thepositions are numbered with reference to SEQ ID NO: 3956. In someembodiments, the polypeptide sequence of the engineeredglycosyltransferase comprises at least one mutation or mutation set atone or more positions selected from Q11D, Q11L, T12I, T12L, F41H, Y44H,Y44S/K187T, L45Y, I55A, I55G, I55L, K56R, K57G, K57H, K57P, N65C, N65G,N65P, N65Q, N65R, S66L, V70W, L72V, P73H, P73S, L74M/M238V, P82S, H83R,H85W, K103H, K103Q, K111F, L113S, Q114G, Q114I, K117N, K117S, Q132G,L135E, G138K, G138S, G138T, P140C, P140G, L159N, K160D, K160Q, K160T,P162L, P167L, R182I, R182L, R182Q, R182T, R182V, R182Y, K214H, K214T,T220G, T220S, L222F, T223D, K226Q, D236L, D236T, D236V, M238A, M238G,M238I, M238R, M238S, M238T, E256R, N286G, E299V, K309P, K309R, K309S,K309T, D387P, D387Q, D387R, E388A, E388S, D389A, D389G, D389L, D389V,R391A, R391L, R391S, R391V, H393P, H393T, T406G, T406S, P408L, P408T,I412G, I412R, R418A, R418H, R418M, K422L, E429D, E430P, Y449G, Y449H,Y449L, Y449R, Y449S, K450N, and K450V, wherein the positions arenumbered with reference to SEQ ID NO: 3956. In some embodiments, thepolypeptide sequence of the engineered glycosyltransferase comprises asequence at least 90% identical to any of SEQ ID NOS: 4148, 4150, 4152,4154, 4156, 4158, 4160, 4162, 4164, 4166, 4168, 4170, 4172, 4174, 4176,4178, 4180, 4182, 4184, 4186, 4188, 4190, 4192, 4194, 4196, 4198, 4200,4202, 4204, 4206, 4208, 4210, 4212, 4214, 4216, 4218, 4220, 4222, 4224,4226, 4228, 4230, 4232, 4234, 4236, 4238, 4240, 4242, 4244, 4246, 4248,4250, 4252, 4254, 4256, 4258, 4260, 4262, 4264, 4266, 4268, 4270, 4272,4274, 4276, 4278, 4280, 4282, 4284, 4286, 4288, 4290, 4292, 4294, 4296,4298, 4300, 4302, 4304, 4306, 4308, 4310, 4312, 4314, 4316, 4318, 4320,4322, 4324, 4326, 4328, 4330, 4332, 4334, 4336, 4338, 4340, 4342, 4344,4346, 4348, 4350, 4352, 4354, 4356, 4358, 4360, 4362, 4364, 4366, 4368,4370, 4372, 4374, 4376, 4378, 4380, 4382, 4384, 4386, 4388, 4390, 4392,4394, 4396, 4398, 4400, 4402, 4404, 4406, 4408, 4410, 4412, 4414, 4416,4418, 4420, 4422, 4424, 4426, 4428, 4430, 4432, 4434, 4436, 4438, 4440,4442, 4444, 4446, 4448, 4450, 4452, 4454, 4456, 4458, 4460, 4462, 4464,4466, 4468, 4470, 4472, 4474, 4476, 4478, 4480, 4482, 4484, 4486, 4488,4490, 4492, 4494, and 4496. In some embodiments, the polypeptidesequence of the engineered glycosyltransferase comprises a sequence atleast 95% identical to any of SEQ ID NOS: 4148, 4150, 4152, 4154, 4156,4158, 4160, 4162, 4164, 4166, 4168, 4170, 4172, 4174, 4176, 4178, 4180,4182, 4184, 4186, 4188, 4190, 4192, 4194, 4196, 4198, 4200, 4202, 4204,4206, 4208, 4210, 4212, 4214, 4216, 4218, 4220, 4222, 4224, 4226, 4228,4230, 4232, 4234, 4236, 4238, 4240, 4242, 4244, 4246, 4248, 4250, 4252,4254, 4256, 4258, 4260, 4262, 4264, 4266, 4268, 4270, 4272, 4274, 4276,4278, 4280, 4282, 4284, 4286, 4288, 4290, 4292, 4294, 4296, 4298, 4300,4302, 4304, 4306, 4308, 4310, 4312, 4314, 4316, 4318, 4320, 4322, 4324,4326, 4328, 4330, 4332, 4334, 4336, 4338, 4340, 4342, 4344, 4346, 4348,4350, 4352, 4354, 4356, 4358, 4360, 4362, 4364, 4366, 4368, 4370, 4372,4374, 4376, 4378, 4380, 4382, 4384, 4386, 4388, 4390, 4392, 4394, 4396,4398, 4400, 4402, 4404, 4406, 4408, 4410, 4412, 4414, 4416, 4418, 4420,4422, 4424, 4426, 4428, 4430, 4432, 4434, 4436, 4438, 4440, 4442, 4444,4446, 4448, 4450, 4452, 4454, 4456, 4458, 4460, 4462, 4464, 4466, 4468,4470, 4472, 4474, 4476, 4478, 4480, 4482, 4484, 4486, 4488, 4490, 4492,4494, and 4496. In some embodiments, the polypeptide sequence of theengineered glycosyltransferase comprises any of SEQ ID NOS: 4148, 4150,4152, 4154, 4156, 4158, 4160, 4162, 4164, 4166, 4168, 4170, 4172, 4174,4176, 4178, 4180, 4182, 4184, 4186, 4188, 4190, 4192, 4194, 4196, 4198,4200, 4202, 4204, 4206, 4208, 4210, 4212, 4214, 4216, 4218, 4220, 4222,4224, 4226, 4228, 4230, 4232, 4234, 4236, 4238, 4240, 4242, 4244, 4246,4248, 4250, 4252, 4254, 4256, 4258, 4260, 4262, 4264, 4266, 4268, 4270,4272, 4274, 4276, 4278, 4280, 4282, 4284, 4286, 4288, 4290, 4292, 4294,4296, 4298, 4300, 4302, 4304, 4306, 4308, 4310, 4312, 4314, 4316, 4318,4320, 4322, 4324, 4326, 4328, 4330, 4332, 4334, 4336, 4338, 4340, 4342,4344, 4346, 4348, 4350, 4352, 4354, 4356, 4358, 4360, 4362, 4364, 4366,4368, 4370, 4372, 4374, 4376, 4378, 4380, 4382, 4384, 4386, 4388, 4390,4392, 4394, 4396, 4398, 4400, 4402, 4404, 4406, 4408, 4410, 4412, 4414,4416, 4418, 4420, 4422, 4424, 4426, 4428, 4430, 4432, 4434, 4436, 4438,4440, 4442, 4444, 4446, 4448, 4450, 4452, 4454, 4456, 4458, 4460, 4462,4464, 4466, 4468, 4470, 4472, 4474, 4476, 4478, 4480, 4482, 4484, 4486,4488, 4490, 4492, 4494, and 4496.

The present invention also provides engineered glycosyltransferases,wherein the polypeptide sequence of the engineered glycosyltransferasescomprise at least one mutation or mutation set at one or more positionsselected from 8/375, 50/137/189/375, 62/153/155/156/159,62/153/155/156/159/427, 62/153/155/199/406, 137/164/375, 153,153/155/156, 153/155/156/159/199/238/406, 153/155/237/238/239/406,153/155/238/239, 153/155/427, 153/156/159, 153/156/199/237/427,153/156/427, 153/159/237/238/352, 153/176/181/427, 155/156,155/156/176/181/199, 155/156/176/238/427, 155/181/199/238/406, 156/199,156/199/427, 164/375/433, 223/375, 375, and 427, wherein the positionsare numbered with reference to SEQ ID NO: 4256. In some embodiments, thepolypeptide sequence of the engineered glycosyltransferase comprises atleast one mutation or mutation set at one or more positions selectedfrom 8V/375L, 50V/137R/189G/375L, 62A/153A/155M/156W/159V,62A/153A/155M/156W/159V/427T, 62A/153A/155M/199S/406Q, 137R/164E/375L,153A, 153A/155M/156W, 153A/155M/156W/159V/199S/238G/406Q,153A/155M/237L/238T/239F/406Q, 153A/155M/238T/239F, 153A/155M/427T,153A/156W/159V, 153A/156W/199S/237L/427T, 153A/156W/427T,153A/159V/237L/238G/352A, 153A/176Q/181L/427S, 155M/156W,155M/156W/176Q/181L/199S, 155M/156W/176Q/238T/427S,155M/181L/199S/238G/406Q, 156W/199S, 156W/199S/427T, 164E/375L/433D,223A/375L, 375L, and 427S, wherein the positions are numbered withreference to SEQ ID NO: 4256. In some embodiments, the polypeptidesequence of the engineered glycosyltransferase comprises at least onemutation or mutation set at one or more positions selected fromS8V/I375L, 150V/Q137R/P189G/I375L, K62A/S153A/F155M/F156W/L159V,K62A/S153A/F155M/F156W/L159V/R427T, K62A/S153A/F155M/N199S/T406Q,Q137R/H164E/I375L, S153A, S153A/F155M/F156W,S153A/F155M/F156W/L159V/N199S/M238G/T406Q,S153A/F155M/P237L/M238T/T239F/T406Q, S153A/F155M/M238T/T239F,S153A/F155M/R427T, S153A/F156W/L159V, S153A/F156W/N199S/P237L/R427T,S153A/F156W/R427T, S153A/L159V/P237L/M238G/S352A,S153A/R176Q/M181L/R427S, F155M/F156W, F155M/F156W/R176Q/M181L/N199S,F155M/F156W/R176Q/M238T/R427S, F155M/M181L/N199S/M238G/T406Q,F156W/N199S, F156W/N199S/R427T, H164E/I375L/N433D, T223A/I375L, I375L,and R427S, wherein the positions are numbered with reference to SEQ IDNO: 4256. In some embodiments, the polypeptide sequence of theengineered glycosyltransferase comprises at least one mutation ormutation set at one or more positions selected from 7, 9, 10, 12, 53,65, 68, 99, 106, 110, 115, 116, 131, 132, 136, 170, 178, 190, 192, 194,200, 220, 238, 242, 245, 257, 272, 280, 302, 304, 335, 385, 395, 399,402, 408, 412, 416, 423, 445, 447, and 449, wherein the positions arenumbered with reference to SEQ ID NO: 4256. In some embodiments, thepolypeptide sequence of the engineered glycosyltransferase comprises atleast one mutation or mutation set at one or more positions selectedfrom 7H, 9G, 10D, 12S, 53N, 65F, 681, 99R, 106K, 106R, 106S, 110G, 115A,115T, 1161, 131C, 131S, 132G, 132R, 132T, 136D, 136G, 136R, 136S, 170E,178L, 190T, 192A, 192L, 192P, 194F, 200S, 220M, 220Q, 220S, 238I, 238L,242L, 245P, 257H, 272H, 280W, 302P, 302S, 304I, 335K, 335R, 385A, 385C,385P, 385S, 395Q, 399K, 402R, 408E, 412R, 416R, 423L, 445H, 445K, 445R,447L, 449K, 449L, 449Q, and 449R, wherein the positions are numberedwith reference to SEQ ID NO: 4256. In some embodiments, the polypeptidesequence of the engineered glycosyltransferase comprises at least onemutation or mutation set at one or more positions selected from G7H,S9G, G10D, T12S, E53N, N65F, H68I, H99R, A106K, A106R, A106S, S110G,N115A, N115T, L116I, E131C, E131S, Q132G, Q132R, Q132T, E136D, E136G,E136R, E136S, A170E, Q178L, D190T, E192A, E192L, E192P, P194F, A200S,T220M, T220Q, T220S, M238I, M238L, I242L, K245P, N257H, R272H, F280W,N302P, N302S, E304I, P335K, P335R, V385A, V385C, V385P, V385S, G395Q,Q399K, K402R, P408E, I412R, K416R, R423L, N445H, N445K, N445R, N447L,Y449K, Y449L, Y449Q, and Y449R, wherein the positions are numbered withreference to SEQ ID NO: 4256. In some embodiments, the polypeptidesequence of the engineered glycosyltransferase comprises a sequence atleast 90% identical to any of SEQ ID NOS: 4498, 4500, 4502, 4504, 4506,4508, 4510, 4512, 4514, 4516, 4518, 4520, 4522, 4524, 4526, 4528, 4530,4532, 4534, 4536, 4538, 4540, 4542, 4544, 4546, 4548, 4550, 4552, 4554,4556, 4558, 4560, 4562, 4564, 4566, 4568, 4570, 4572, 4574, 4576, 4578,4580, 4582, 4584, 4586, 4588, 4590, 4592, 4594, 4596, 4598, 4600, 4602,4604, 4606, 4608, 4610, 4612, 4614, 4616, 4618, 4620, 4622, 4624, 4626,4628, 4630, 4632, 4634, 4636, 4638, 4640, 4642, 4644, 4646, 4648, 4650,4652, 4654, 4656, 4658, 4660, 4662, 4664, 46664668, 4670, 4672, 4674,4676, 4678, 4680, and 4682. In some embodiments, the polypeptidesequence of the engineered glycosyltransferase comprises a sequence atleast 95% identical to any of SEQ ID NOS: 4498, 4500, 4502, 4504, 4506,4508, 4510, 4512, 4514, 4516, 4518, 4520, 4522, 4524, 4526, 4528, 4530,4532, 4534, 4536, 4538, 4540, 4542, 4544, 4546, 4548, 4550, 4552, 4554,4556, 4558, 4560, 4562, 4564, 4566, 4568, 4570, 4572, 4574, 4576, 4578,4580, 4582, 4584, 4586, 4588, 4590, 4592, 4594, 4596, 4598, 4600, 4602,4604, 4606, 4608, 4610, 4612, 4614, 4616, 4618, 4620, 4622, 4624, 4626,4628, 4630, 4632, 4634, 4636, 4638, 4640, 4642, 4644, 4646, 4648, 4650,4652, 4654, 4656, 4658, 4660, 4662, 4664, 4666 4668, 4670, 4672, 4674,4676, 4678, 4680, and 4682. In some embodiments, the polypeptidesequence of the engineered glycosyltransferase comprises any of SEQ IDNOS: 4498, 4500, 4502, 4504, 4506, 4508, 4510, 4512, 4514, 4516, 4518,4520, 4522, 4524, 4526, 4528, 4530, 4532, 4534, 4536, 4538, 4540, 4542,4544, 4546, 4548, 4550, 4552, 4554, 4556, 4558, 4560, 4562, 4564, 4566,4568, 4570, 4572, 4574, 4576, 4578, 4580, 4582, 4584, 4586, 4588, 4590,4592, 4594, 4596, 4598, 4600, 4602, 4604, 4606, 4608, 4610, 4612, 4614,4616, 4618, 4620, 4622, 4624, 4626, 4628, 4630, 4632, 4634, 4636, 4638,4640, 4642, 4644, 4646, 4648, 4650, 4652, 4654, 4656, 4658, 4660, 4662,4664, 46664668, 4670, 4672, 4674, 4676, 4678, 4680, and 4682.

The present invention also provides engineered glycosyltransferases,wherein the polypeptide sequence of the engineered glycosyltransferasescomprise at least one mutation or mutation set at one or more positionsselected from 11/41/44/127/132/406, 11/41/44/127/278/406,11/41/45/73/127/278/406/408, 11/41/45/127/278/406, 11/41/132/278,11/44/45/127, 11/44/73/127/132/135, 11/44/73/127/132/135/406/408,11/44/127, 11/44/132/278/406, 11/45/73/127/135,11/45/127/132/135/138/182, 11/45/406, 11/73/127/132/135/182/278,11/73/127/132/278/406/408, 11/73/127/132/406/408, 11/73/132,11/73/132/135/408, 11/127/132/135, 11/127/132/135/182,11/127/132/135/406, 11/127/132/138/182, 11/127/132/182, 11/132/135/406,11/182/406, 41/44/45/73/127/132/278, 41/45/127/132/135/278/406,41/73/132/135/406, 44/45/73/132/135/406,44/45/127/132/135/138/182/406/408, 44/45/127/132/278, 44/73/127/135,44/73/127/135/182/278/406/408, 44/73/132/135/406/408,45/73/127/132/135/406/408, 45/73/132, 45/73/132/135/182,45/73/278/406/408, 45/127/132, 45/127/132/135/182,45/127/132/135/182/406/408, 45/127/135, 45/132/408, 45/406, 56/309/449,57, 65/114/422/427, 65/114/427, 65/143, 65/143/235/427, 73/127,73/127/132, 127, 127/132/135/182, 127/132/135/182/406, 132/135,132/135/182, 132/135/406, 132/406, 164, 164/220/309/449, 164/220/449,164/449, 182, 220/449, 309, 309/449, 406, 418/427, 427, 427/429, and449, wherein the positions are numbered with reference to SEQ ID NO:4550. In some embodiments, the polypeptide sequence of the engineeredglycosyltransferase comprises at least one mutation or mutation set atone or more positions selected from 11L/41H/44H/127I/132K/406G,11L/41H/44H/127I/278L/406S, 11L/41H/45Y/73H/127I/278L/406G/408A,11L/41H/45Y/127I/278L/406G, 11L/41H/132K/278L, 11L/44H/45Y/127I,11L/44H/73H/127I/132K/135E, 11L/44H/73S/127I/132K/135E/406G/408A,11L/44H/127I, 11L/44H/132K/278L/406G, 11L/45Y/73H/127I/135E,11L/45Y/127I/132K/135E/138T/182T, 11L/45Y/406G,11L/73H/127I/132K/278L/406G/408A, 11L/73H/127I/132K/406G/408A,11L/73H/132K, 11L/73S/127I/132K/135E/182L/278L, 11L/73S/132K/135E/408A,11L/127I/132K/135E, 11L/127I/132K/135E/182L, 11L/127I/132K/135E/406S,11L/127I/132K/138T/182L, 11L/127I/132K/182T, 11L/132K/135E/406G,11L/182L/406S, 41H/44H/45Y/73H/127I/132K/278L,41H/45Y/127I/132K/135E/278L/406S, 41H/73H/132K/135E/406G,44H/45Y/73H/132K/135E/406G, 44H/45Y/1271132K/135E/138T/182L/406S/408A,44H/45Y/1271/132K/278L, 44H/73H/1271/135E,44H/73S/127I/135E/182L/278L/406G/408A, 44H/73S/132K/135E/406G/408A,45Y/73H/127I/132K/135E/406G/408A, 45Y/73H/132K, 45Y/73H/132K/135E/182L,45Y/73S/278L/406G/408A, 45Y/127I/132K, 45Y/127I/132K/135E/182L,45Y/127I/132K/135E/182T/406G/408A, 45Y/127I/135E, 45Y/132K/408A,45Y/406G, 56R/309P/449L, 57G, 65G/114I/422L/427S, 65G/114I/427S,65G/143K, 65G/143K/235R/427S, 73H/127I, 73H/127I/132K, 127I,127I/132K/135E/182I/406G, 127I/132K/135E/182L, 132K/135E,132K/135E/182I, 132K/135E/406G, 132K/406S, 164E, 164E/220G/309S/449H,164E/220G/449L, 164E/449H, 182L, 220G/449H, 309P/449L, 309R, 309T/449H,406G, 418A/427S, 427S, 427S/429D, and 449L, wherein the positions arenumbered with reference to SEQ ID NO: 4550. In some embodiments, thepolypeptide sequence of the engineered glycosyltransferase comprises atleast one mutation or mutation set at one or more positions selectedfrom Q11L/F41H/Y44H/Q127I/Q132K/T406G, Q11L/F41H/Y44H/Q127I/V278L/T406S,Q11L/F41H/L45Y/A73H/Q127I/V278L/T406G/P408A,Q11L/F41H/L45Y/Q127I/V278L/T406G, Q11L/F41H/Q132K/V278L,Q11L/Y44H/L45Y/Q127I, Q11L/Y44H/A73H/Q127I/Q132K/L135E,Q11L/Y44H/A73S/Q127I/Q132K/L135E/T406G/P408A, Q11L/Y44H/Q127I,Q11L/Y44H/Q132K/V278L/T406G, Q11L/L45Y/A73H/Q127I/L135E,Q11L/L45Y/Q127I/Q132K/L135E/G138T/R182T, Q11L/L45Y/T406G,QIL/A73H/Q127I/Q132K/V278L/T406G/P408A,Q11L/A73H/Q27I/Q132K/T406G/P408A, Q11L/A73H/Q132K,Q11L/A73S/Q127I/Q132K/L135E/R182L/V278L, Q11L/A73S/Q132K/L135E/P408A,Q11L/Q127I/Q132K/L135E, Q11L/Q127I/Q132K/L135E/R182L,Q11L/Q127I/Q132K/L135E/T406S, Q11L/Q127I/Q132K/G138T/R182L,Q11L/Q127I/Q132K/R182T, Q11L/Q132K/L135E/T406G, Q11L/R182L/T406S,F41H/Y44H/L45Y/A73H/Q127I/Q132K/V278L,F41H/L45Y/Q127I/Q132K/L135E/V278L/T406S, F41H/A73H/Q132K/L135E/T406G,Y44H/L45Y/A73H/Q132K/L135E/T406G,Y44H/L45Y/Q127I/Q132K/L135E/G138T/R182L/T406S/P408A,Y44H/L45Y/Q127I/Q132K/V278L, Y44H/A73H/Q127I/L135E,Y44H/A73S/Q127I/L135E/R182L/V278L/T406G/P408A,Y44H/A73S/Q132K/L135E/T406G/P408A,L45Y/A73H/Q127I/Q132K/L135E/T406G/P408A, L45Y/A73H/Q132K,L45Y/A73H/Q132K/L135E/R182L, L45Y/A73S/V278L/T406G/P408A,L45Y/Q127I/Q132K, L45Y/Q127I/Q132K/L135E/R182L,L45Y/Q127I/Q132K/L135E/R182T/T406G/P408A, L45Y/Q127I/L135E,L45Y/Q132K/P408A, L45Y/T406G, K56R/K309P/Y449L, K57G,N65G/Q114I/K422L/R427S, N65G/Q14I/R427S, N65G/P143K,N65G/P143K/Q235R/R427S, A73H/Q127I, A73H/Q127I/Q132K, Q127I,Q127I/Q132K/L135E/R182I/T406G, Q127I/Q132K/L135E/R182L, Q132K/L135E,Q132K/L135E/R182I, Q132K/L135E/T406G, Q132K/T406S, H164E,H164E/T220G/K309S/Y449H, H164E/T220G/Y449L, H164E/Y449H, R182L,T220G/Y449H, K309P/Y449L, K309R, K309T/Y449H, T406G, R418A/R427S, R427S,R427S/E429D, and Y449L, wherein the positions are numbered withreference to SEQ ID NO: 4550. In some embodiments, the polypeptidesequence of the engineered glycosyltransferase comprises a sequence atleast 90% identical to any of SEQ ID NOS: 7216, 7218, 7220, 7222, 7224,7226, 7228, 7230, 7232, 7234, 7236, 7238, 7240, 7242, 7244, 7246, 7248,7250, 7252, 7254, 7256, 7258, 7260, 7262, 7264, 7266, 7268, 7270, 7272,7274, 7276, 7278, 7280, 7282, 7284, 7286, 7288, 7290, 7292, 7294, 7296,7298, 7300, 7302, 7304, 7306, 7308, 7310, 7312, 7314, 7316, 7318, 7320,7322, 7324, 7326, 7328, 7330, 7332, 7334, 7336, 7338, 7340, 7342, 7344,7346, 7348, 7350, 7352, 7354, 7356, 7358, and 7360. In some embodiments,the polypeptide sequence of the engineered glycosyltransferase comprisesa sequence at least 95% identical to any of SEQ ID NOS: 7216, 7218,7220, 7222, 7224, 7226, 7228, 7230, 7232, 7234, 7236, 7238, 7240, 7242,7244, 7246, 7248, 7250, 7252, 7254, 7256, 7258, 7260, 7262, 7264, 7266,7268, 7270, 7272, 7274, 7276, 7278, 7280, 7282, 7284, 7286, 7288, 7290,7292, 7294, 7296, 7298, 7300, 7302, 7304, 7306, 7308, 7310, 7312, 7314,7316, 7318, 7320, 7322, 7324, 7326, 7328, 7330, 7332, 7334, 7336, 7338,7340, 7342, 7344, 7346, 7348, 7350, 7352, 7354, 7356, 7358, and 7360. Insome embodiments, the polypeptide sequence of the engineeredglycosyltransferase comprises any of SEQ ID NOS: 7216, 7218, 7220, 7222,7224, 7226, 7228, 7230, 7232, 7234, 7236, 7238, 7240, 7242, 7244, 7246,7248, 7250, 7252, 7254, 7256, 7258, 7260, 7262, 7264, 7266, 7268, 7270,7272, 7274, 7276, 7278, 7280, 7282, 7284, 7286, 7288, 7290, 7292, 7294,7296, 7298, 7300, 7302, 7304, 7306, 7308, 7310, 7312, 7314, 7316, 7318,7320, 7322, 7324, 7326, 7328, 7330, 7332, 7334, 7336, 7338, 7340, 7342,7344, 7346, 7348, 7350, 7352, 7354, 7356, 7358, and 7360.

The present invention also provides engineered glycosyltransferases,wherein the polypeptide sequence of the engineered glycosyltransferasescomprise at least one mutation or mutation set at one or more positionsselected from 7/10/73/127/132/385/423, 10, 10/53/73/99,10/53/272/423/427, 10/53/423, 10/73, 10/73/127, 10/127/132/385,10/132/302/385, 53/73/200/423, 53/127/132/385, 53/127/136/385,53/132/136/302/423/427, 53/132/302/385/423/427, 65/106/445/447/449,65/143/220, 65/220/309/445/447, 65/220/445/449, 65/399/406/447/449,65/402/406/445/449, 65/445/447/449, 65/447/449, 73, 73/127/427,73/132/136/385/427, 73/385, 73/385/427, 106/220/399/402/406,115/116/278, 115/170/190/238/412, 115/190/194, 115/245/278, 116/170/190,116/190/406/408, 116/238, 116/238/245, 116/416, 127, 132,143/220/445/447, 143/309/402, 143/309/445/447/449, 170, 170/190/192/194,170/192, 170/192/194/278, 170/192/194/335, 170/194, 170/194/335/416,170/238, 170/335, 170/335/416, 190, 190/192/194, 190/194,190/194/238/245, 190/194/335/416, 190/194/412, 190/245/412, 192/194,192/194/242/406/408, 194, 200, 200/385, 220/399, 220/445, 220/445/447,257/385, 272/302, 272/385, 278, 302/385, 309/399/449, 309/445/447/449,385, 385/427, 399/406, 399/406/449, 402/445/449, 406/445/447/449,406/445/449, 423/427, 445/447/449, 445/449, 447, and 449, wherein thepositions are numbered with reference to SEQ ID NO: 7324. In someembodiments, the polypeptide sequence of the engineeredglycosyltransferase comprises at least one mutation or mutation set atone or more positions selected from 7V/10D/73H/127I/132G/385P/423L, 10D,10D/53N/73H/99R, 10D/53N/272H/423L/427S, 10D/53N/423L, 10D/73H,10D/73H/127I, 10D/127I/132R/385P, 10D/132R/302P/385P, 53N/73H/200S/423L,53N/127I/132R/385S, 53N/127I/136R/385L, 53N/132G/136R/302P/423L/427S,53N/132G/302P/385S/423L/427S, 65G/106R/445K/447L/449L, 65G/143K/220Q,65G/220Q/309P/445R/447L, 65G/220Q/445R/449K, 65G/220Q/445R/449R,65G/399K/406G/447L/449R, 65G/402R/406G/445R/449R, 65G/445K/447L/449R,65G/447L/449L, 73H, 73H/127I/427S, 73H/132R/136R/385S/427S, 73H/385S,73H/385S/427S, 106R/220Q/399K/402R/406G, 115A/170E/190T/238L/412R,115A/190T/194F, 115A/245P/278L, 115T/116I/278L, 116I/170E/190T,116I/190T/406G/408A, 116I/238L, 116I/238L/245P, 116I/416R, 1271, 132G,143K/220Q/445K/447L, 143K/309P/402R, 143K/309P/445K/447L/449L, 170E,170E/190T/192L/194F, 170E/192L, 170E/192L/194F/278L,170E/192L/194F/335K, 170E/194F, 170E/194F/335R/416R, 170E/238L,170E/335K, 170E/335K/416R, 190T, 190T/192L/194F, 190T/194F,190T/194F/238L/245P, 190T/194F/335R/416R, 190T/194F/412R,190T/245P/412R, 192L/194F, 192L/194F/242L/406G/408A, 194F, 200S,200S/385S, 220M/445K, 220Q/399K, 220Q/445K/447L, 257H/385S, 272H/302P,272H/385P, 278L, 302P/385P, 302P/385S, 309P/399K/449L,309P/445K/447L/449L, 385S, 385S/427S, 399K/406G, 399K/406G/449L,402R/445R/449L, 406G/445K/449R, 406G/445R/447L/449L, 406G/445R/449K,423L/427S, 445K/449L, 445R/447L/449L, 445R/449L, 447L, and 449L, whereinthe positions are numbered with reference to SEQ ID NO: 7324. In someembodiments, the polypeptide sequence of the engineeredglycosyltransferase comprises at least one mutation or mutation set atone or more positions selected fromG7V/G10D/A73H/Q127I/K132G/V385P/R423L, G10D, G10D/E53N/A73H/H99R,G10D/E53N/R272H/R423L/R427S, G10D/E53N/R423L, G10D/A73H,G10D/A73H/Q127I, G10D/Q127I/K132R/V385P, G10D/K132R/N302P/V385P,E53N/A73H/A200S/R423L, E53N/Q127I/K132R/V385S, E53N/Q127I/E136R/V385L,E53N/K132G/E136R/N302P/R423L/R427S, E53N/K132G/N302P/V385S/R423L/R427S,N65G/A106R/N445K/N447L/Y449L, N65G/P143K/T220Q,N65G/T220Q/K309P/N445R/N447L, N65G/T220Q/N445R/Y449K,N65G/T220Q/N445R/Y449R, N65G/Q399K/S406G/N447L/Y449R,N65G/K402R/S406G/N445R/Y449R, N65G/N445K/N447L/Y449R, N65G/N447L/Y449L,A73H, A73H/Q127I/R427S, A73H/K132R/E136R/V385S/R427S, A73H/V385S,A73H/V385S/R427S, A106R/T220Q/Q399K/K402R/S406G,N115A/A170E/D190T/M238L/I412R, N115A/D190T/P194F, N115A/K245P/V278L,N115T/L116I/V278L, L16I/A170E/D190T, L16I/D190T/S406G/P408A,L116I/M238L, L116I/M238L/K245P, L116I/K416R, Q127I, K132G,P143K/T220Q/N445K/N447L, P143K/K309P/K402R,P143K/K309P/N445K/N447L/Y449L, A170E, A170E/D190T/E192L/P194F,A170E/E192L, A170E/E192L/P194F/V278L, A170E/E192L/P194F/P335K,A170E/P194F, A170E/P194F/P335R/K416R, A170E/M238L, A170E/P335K,A170E/P335K/K416R, D190T, D190T/E192L/P194F, D190T/P194F,D190T/P194F/M238L/K245P, D190T/P194F/P335R/K416R, D190T/P194F/I412R,D190T/K245P/I412R, E192L/P194F, E192L/P194F/I242L/S406G/P408A, P194F,A200S, A200S/V385S, T220M/N445K, T220Q/Q399K, T220Q/N445K/N447L,N257H/V385S, R272H/N302P, R272H/V385P, V278L, N302P/V385P, N302P/V385S,K309P/Q399K/Y449L, K309P/N445K/N447L/Y449L, V385S, V385S/R427S,Q399K/S406G, Q399K/S406G/Y449L, K402R/N445R/Y449L, S406G/N445K/Y449R,S406G/N445R/N447L/Y449L, S406G/N445R/Y449K, R423L/R427S, N445K/Y449L,N445R/N447L/Y449L, N445R/Y449L, N447L, and Y449L, wherein the positionsare numbered with reference to SEQ ID NO: 7324. In some embodiments, thepolypeptide sequence of the engineered glycosyltransferase comprises atleast one mutation or mutation set at one or more positions selectedfrom 2, 4, 11/44, 11/71, 11/287, 11/441, 37, 40, 42, 42/138, 42/141, 43,46, 47, 48/398, 49, 49/406/408, 51, 64, 71, 76, 97, 100, 108, 108/172,109, 112, 117/157/301, 118, 118/406/408, 119, 119/172, 130, 133, 134,141, 157, 169, 172/420, 172/437, 179, 181, 259, 274, 275, 287, 288,333/398, 333/406/408, 338, 356, 357, 376, 381, 385, 394/420, 396,397/406/408, 398, 401, 406/408, 410, 417, 420, 434, 437, and 441,wherein the positions are numbered with reference to SEQ ID NO: 7324. Insome embodiments, the polypeptide sequence of the engineeredglycosyltransferase comprises at least one mutation or mutation set atone or more positions selected from 2T, 4F, 11L/44H, 11L/71P, 11L/71T,11L/287S, 11L/441S, 37G, 40Q, 42A, 42A/141P, 42D/138C, 42G, 43A, 43L,43M, 43T, 46C, 46L, 46Q, 46T, 47N, 48S/398V, 49G, 49K, 49N/406G/408A,51M, 51Q, 51V, 64A, 64F, 64G, 64L, 64Q, 64R, 64V, 71T, 76H, 76L, 97L,97V, 100R, 108G/172H, 108T, 109L, 109V, 109Y, 112N, 112Q, 112R, 112T,112V, 117R/157T/301R, 118A, 118A/406G/408A, 1181, 118L, 1185, 118V,119A, 119G, 1195, 119T/172H, 130S, 133H, 133L, 133R, 1335, 1345, 141G,141Q, 1415, 157C, 157E, 157T, 169E, 172H/420L, 172H/437D, 1791, 179R,181H, 181T, 259T, 274Q, 274T, 275L, 287A, 287K, 287L, 2875, 288P,333S/398T, 333S/406G/408A, 338S, 356S, 356T, 357T, 376G, 376M, 376N,376R, 3765, 381C, 385A, 394H/420W, 396A, 3965, 397L/406G/408A, 398H,398K, 398L, 398M, 398R, 401T, 406G/408A, 410R, 417C, 4175, 420F, 420L,434C, 437G, 437V, and 441L, wherein the positions are numbered withreference to SEQ ID NO: 7324. In some embodiments, the polypeptidesequence of the engineered glycosyltransferase comprises at least onemutation or mutation set at one or more positions selected from H2T,H4^(F), QIL/Y44H, Q11L/E71P, Q11L/E71T, Q11L/V287S, Q11L/Q441S, A37G,G40Q, V42A, V42A/A141P, V42D/G138C, V42G, I43A, I43L, I43M, I43T, V46C,V46L, V46Q, V46T, S47N, T48S/A398V, A49G, A49K, A49N/S406G/P408A, N51M,N51Q, N51V, S64A, S64F, S64G, S64L, S64Q, S64R, S64V, E71T, E76H, E76L,T97L, T97V, K100R, N108G/R172H, N108T, F109L, F109V, F109Y, 1112N,1112Q, 1112R, 1112T, 1112V, K117R/N157T/Q301R, P118A, P118A/S406G/P408A,P1181, P118L, P118S, P118V, D119A, D119G, D119S, D119T/R172H, A130S,V133H, V133L, V133R, V133S, A134S, A141G, A141Q, A141S, N157C, N157E,N157T, P169E, R172H/I420L, R172H/E437D, D179I, D179R, M181H, M181T,V259T, D274Q, D274T, M275L, V287A, V287K, V287L, V287S, N288P,N333S/A398T, N333S/S406G/P408A, G338S, G356S, G356T, V357T, V376G,V376M, V376N, V376R, V376S, A381C, V385A, R394H/I420W, E396A, E396S,I397L/S406G/P408A, A398H, A398K, A398L, A398M, A398R, L401T,S406G/P408A, G410R, V417C, V417S, I420F, I420L, A434C, E437G, E437V, andQ441L, wherein the positions are numbered with reference to SEQ ID NO:7324. In some embodiments, the polypeptide sequence of the engineeredglycosyltransferase comprises a sequence at least 90% identical to anyof SEQ ID NOS: 7766, 7768, 7770, 7772, 7774, 7776, 7778, 7780, 7782,7784, 7786, 7788, 7790, 7792, 7794, 7796, 7798, 7800, 7812, 7814, 7816,7818, 7820, 7822, 7824, 7826, 7828, 7830, 7832, 7834, 7836, 7838, 7840,7842, 7844, 7846, 7848, 7850, 7852, 7854, 7856, 7858, 7860, 7862, 7864,7866, 7868, 7870, 7872, 7874, 7876, 7878, 7880, 7882, 7884, 7886, 7888,7890, 7892, 7894, 7896, 7898, 7900, 7902, 7904, 7906, 7908, 7910, 7912,7914, 7916, 7918, 7920, 7922, 7924, 7926, 7928, 7930, 7932, 7934, 7936,7938, 7940, 7942, 7944, 8092, 8094, 8096, 8098, 8100, 8102, 8104, 8106,8108, 8110, 8112, 8114, 8116, 8118, 8120, 8122, 8124, 8126, 8128, 8130,8132, 8134, 8136, 8138, 8140, 8142, 8144, 8146, 8148, 8150, 8152, 8154,8156, 8158, 8160, 8162, 8164, 8166, 8168, 8170, 8172, 8174, 8176, 8178,8180, 8182, 8184, 8186, 8188, 8190, 8192, 8194, 8196, 8198, 8200, 8202,8204, 8206, 8208, 8210, 8212, 8214, 8216, 8218, 8220, 8222, 8224, 8226,8228, 8230, 8232, 8234, 8236, 8238, 8240, 8242, 8244, 8246, 8248, 8250,8252, 8254, 8256, 8258, 8260, 8262, 8264, 8266, 8268, 8270, 8272, 8274,8276, 8278, 8280, 8282, 8284, 8286, 8288, 8290, 8292, 8294, 8296, 8298,8300, 8302, 8304, 8306, 8308, 8310, 8312, 8314, 8316, 8318, 8320, 8322,8324, 8326, 8328, 8330, 8332, 8334, 8336, and 8338. In some embodiments,the polypeptide sequence of the engineered glycosyltransferase comprisesa sequence at least 95% identical to any of SEQ ID NOS: 7766, 7768,7770, 7772, 7774, 7776, 7778, 7780, 7782, 7784, 7786, 7788, 7790, 7792,7794, 7796, 7798, 7800, 7812, 7814, 7816, 7818, 7820, 7822, 7824, 7826,7828, 7830, 7832, 7834, 7836, 7838, 7840, 7842, 7844, 7846, 7848, 7850,7852, 7854, 7856, 7858, 7860, 7862, 7864, 7866, 7868, 7870, 7872, 7874,7876, 7878, 7880, 7882, 7884, 7886, 7888, 7890, 7892, 7894, 7896, 7898,7900, 7902, 7904, 7906, 7908, 7910, 7912, 7914, 7916, 7918, 7920, 7922,7924, 7926, 7928, 7930, 7932, 7934, 7936, 7938, 7940, 7942, 7944, 8092,8094, 8096, 8098, 8100, 8102, 8104, 8106, 8108, 8110, 8112, 8114, 8116,8118, 8120, 8122, 8124, 8126, 8128, 8130, 8132, 8134, 8136, 8138, 8140,8142, 8144, 8146, 8148, 8150, 8152, 8154, 8156, 8158, 8160, 8162, 8164,8166, 8168, 8170, 8172, 8174, 8176, 8178, 8180, 8182, 8184, 8186, 8188,8190, 8192, 8194, 8196, 8198, 8200, 8202, 8204, 8206, 8208, 8210, 8212,8214, 8216, 8218, 8220, 8222, 8224, 8226, 8228, 8230, 8232, 8234, 8236,8238, 8240, 8242, 8244, 8246, 8248, 8250, 8252, 8254, 8256, 8258, 8260,8262, 8264, 8266, 8268, 8270, 8272, 8274, 8276, 8278, 8280, 8282, 8284,8286, 8288, 8290, 8292, 8294, 8296, 8298, 8300, 8302, 8304, 8306, 8308,8310, 8312, 8314, 8316, 8318, 8320, 8322, 8324, 8326, 8328, 8330, 8332,8334, 8336, and 8338. In some embodiments, the polypeptide sequence ofthe engineered glycosyltransferase comprises any of SEQ ID NOS: 7766,7768, 7770, 7772, 7774, 7776, 7778, 7780, 7782, 7784, 7786, 7788, 7790,7792, 7794, 7796, 7798, 7800, 7812, 7814, 7816, 7818, 7820, 7822, 7824,7826, 7828, 7830, 7832, 7834, 7836, 7838, 7840, 7842, 7844, 7846, 7848,7850, 7852, 7854, 7856, 7858, 7860, 7862, 7864, 7866, 7868, 7870, 7872,7874, 7876, 7878, 7880, 7882, 7884, 7886, 7888, 7890, 7892, 7894, 7896,7898, 7900, 7902, 7904, 7906, 7908, 7910, 7912, 7914, 7916, 7918, 7920,7922, 7924, 7926, 7928, 7930, 7932, 7934, 7936, 7938, 7940, 7942, 7944,8092, 8094, 8096, 8098, 8100, 8102, 8104, 8106, 8108, 8110, 8112, 8114,8116, 8118, 8120, 8122, 8124, 8126, 8128, 8130, 8132, 8134, 8136, 8138,8140, 8142, 8144, 8146, 8148, 8150, 8152, 8154, 8156, 8158, 8160, 8162,8164, 8166, 8168, 8170, 8172, 8174, 8176, 8178, 8180, 8182, 8184, 8186,8188, 8190, 8192, 8194, 8196, 8198, 8200, 8202, 8204, 8206, 8208, 8210,8212, 8214, 8216, 8218, 8220, 8222, 8224, 8226, 8228, 8230, 8232, 8234,8236, 8238, 8240, 8242, 8244, 8246, 8248, 8250, 8252, 8254, 8256, 8258,8260, 8262, 8264, 8266, 8268, 8270, 8272, 8274, 8276, 8278, 8280, 8282,8284, 8286, 8288, 8290, 8292, 8294, 8296, 8298, 8300, 8302, 8304, 8306,8308, 8310, 8312, 8314, 8316, 8318, 8320, 8322, 8324, 8326, 8328, 8330,8332, 8334, 8336, and 8338.

The present invention also provides engineered glycosyltransferases,wherein the polypeptide sequence of the engineered glycosyltransferasescomprise at least one mutation or mutation set at one or more positionsselected from 11, 11/64/109, 11/445, 42/43, 42/44/71/73/116, 43/73/141,46/47/51, 46/51, 47/49, 47/51, 64, 64/65/109, 64/65/112, 64/112, 64/134,64/445, 65/112, 65/112/445, 71, 71/73, 71/73/141, 71/141, 71/302, 73,73/116/141, 73/141, 73/302, 109, 109/112, 109/115/118, 109/134, 109/406,112, 112/445, 116/287, 127, 127/169/172, 127/169/287,127/169/376/398/399, 127/169/398/399, 127/287, 127/376, 141, 141/302,169/172/287, 169/172/288, 169/172/288/398/399/420/423/427,169/172/398/399, 169/287, 169/398, 169/398/399, 287, 287/288, 287/376,287/398, 287/399, 287/420/423/427, 288, 288/376/398, 288/398, 288/399,302, 376, 376/398, 376/399, 398, 398/399, 398/399/420, 398/427, 399, and420, wherein the positions are numbered with reference to SEQ ID NO:7784. In some embodiments, the polypeptide sequence of the engineeredglycosyltransferase comprises at least one mutation or mutation set atone or more positions selected from 11L, 11L/64R/109V, 11L/445K,42A/44H/71T/73H/116I, 42G/43M, 43T/73H/141P, 46L/47N/51Q, 46T/51Q,47N/49G, 47N/51V, 64R, 64R/65G/109V, 64R/65G/112N, 64R/65G/112T,64R/112N, 64R/134S, 64R/445K, 65G/112N, 65G/112N/445K, 71T, 71T/73H,71T/73H/141G, 71T/73H/141S, 71T/141P, 71T/302P, 73H, 73H/116I/141P,73H/141G, 73H/141P, 73H/141S, 73H/302P, 109L/112T, 109V, 109V/112T,109V/115A/118V, 109V/134S, 109V/406G, 112N/445K, 112T, 116I/287S, 127I,127I/169E/172H, 127I/169E/287S, 127I/169E/376M/398M/399K,127I/169E/398L/399K, 127I/287S, 127I/376M, 141G, 141G/302P, 141P, 141S,141S/302P, 169E/172H/287S, 169E/172H/288P,169E/172H/288P/398L/399K/420F/423L/427S, 169E/172H/398M/399K, 169E/287S,169E/398M/399K, 169E/398T, 287L/288P, 287L/399K, 287M, 287S, 287S/376M,287S/398T, 2875/399K, 287S/420F/423L/427S, 288P, 288P/376S/398L,288P/398K, 288P/399K, 302P, 376M, 376M/398L, 376M/398M, 376M/399K, 398L,398L/399K, 398L/427S, 398M, 398M/399K, 398M/399K/420F, 398R, 398R/399K,398T/399K, 399K, and 420F, wherein the positions are numbered withreference to SEQ ID NO: 7784. In some embodiments, the polypeptidesequence of the engineered glycosyltransferase comprises at least onemutation or mutation set at one or more positions selected from Q11L,Q11L/S64R/F109V, Q11L/N445K, V42A/Y44H/E71T/A73H/L116I, V42G/I43M,I43T/A73H/A141P, V46L/S47N/N51Q, V46T/N51Q, S47N/A49G, S47N/N51V, S64R,S64R/N65G/F109V, S64R/N65G/I112N, S64R/N65G/I112T, S64R/I112N,S64R/A134S, S64R/N445K, N65G/I112N, N65G/I112N/N445K, E71T, E71T/A73H,E71T/A73H/A141G, E71T/A73H/A141S, E71T/A141P, E71T/N302P, A73H,A73H/L116I/A141P, A73H/A141G, A73H/A141P, A73H/A141S, A73H/N302P,F109L/I112T, F109V, F109V/I112T, F109V/N115A/P118V, F109V/A134S,F109V/S406G, I112N/N445K, 1112T, L116I/V287S, Q127I, Q127I/P169E/R172H,Q127I/P169E/V287S, Q127I/P169E/V376M/A398M/Q399K,Q127I/P169E/A398L/Q399K, Q127I/V287S, Q127I/V376M, A141G, A141G/N302P,A141P, A141S, A141S/N302P, P169E/R172H/V287S, P169E/R172H/N288P,P169E/R172H/N288P/A398L/Q399K/I420F/R423L/R427S,P169E/R172H/A398M/Q399K, P169E/V287S, P169E/A398M/Q399K, P169E/A398T,V287L/N288P, V287L/Q399K, V287M, V287S, V287S/V376M, V287S/A398T,V287S/Q399K, V287S/I420F/R423L/R427S, N288P, N288P/V376S/A398L,N288P/A398K, N288P/Q399K, N302P, V376M, V376M/A398L, V376M/A398M,V376M/Q399K, A398L, A398L/Q399K, A398L/R427S, A398M, A398M/Q399K,A398M/Q399K/I420F, A398R, A398R/Q399K, A398T/Q399K, Q399K, and I420F,wherein the positions are numbered with reference to SEQ ID NO: 7784. Insome embodiments, the polypeptide sequence of the engineeredglycosyltransferase comprises at least one mutation or mutation set atone or more positions selected from 10, 10/144, 10/199, 13, 14, 15,15/394, 16, 22, 36, 89, 93, 96, 116, 116/123, 116/143, 116/350, 123,125, 127, 143, 144, 149, 156, 186, 187, 197, 198, 199, 201, 202, 203,268, 287, 324, 331, and 350, wherein the positions are numbered withreference to SEQ ID NO: 7784. In some embodiments, the polypeptidesequence of the engineered glycosyltransferase comprises at least onemutation or mutation set at one or more positions selected from 10D,10D/144L, 10D/199G, 13Q, 14Q, 15A, 15L/394H, 16A, 16G, 16T, 22G, 36M,89A, 93A, 96M, 1161, 116I/123N, 116I/123S, 116I/143R, 116I/350L, 123A,123G, 123L, 123S, 123V, 125L, 127A, 127G, 127V, 143G, 144G, 144Q, 144S,149S, 156V, 186N, 187G, 187Y, 197H, 198Q, 199G, 199P, 199R, 199S, 199Y,201A, 201K, 201L, 201N, 202A, 202Y, 203T, 268W, 287A, 324R, 331C, 331V,and 350L, wherein the positions are numbered with reference to SEQ IDNO: 7784. In some embodiments, the polypeptide sequence of theengineered glycosyltransferase comprises at least one mutation ormutation set at one or more positions selected from G10D, G10D/V144L,G10D/N199G, L13Q, R14Q, V15A, V15L/R394H, L16A, L16G, L16T, A22G, L36M,G89A, H93A, G96M, L116I, L116I/Y123N, L116I/Y123S, L116I/P143R,L116I/M350L, Y123A, Y123G, Y123L, Y123S, Y123V, F125L, Q127A, Q127G,Q127V, P143G, V144G, V144Q, V144S, A149S, F156V, G186N, T187G, T187Y,P197H, G198Q, N199G, N199P, N199R, N199S, N199Y, G201A, G201K, G201L,G201N, I202A, I202Y, M203T, Y268W, V287A, H324R, 1331C, 1331V, andM350L, wherein the positions are numbered with reference to SEQ ID NO:7784. In some embodiments, the polypeptide sequence of the engineeredglycosyltransferase comprises a sequence at least 90% identical to anyof SEQ ID NOS: 8624, 8626, 8628, 8630, 8632, 8634, 8636, 8638, 8640,8642, 8644, 8646, 8648, 8650, 8652, 8654, 8656, 8658, 8660, 8662, 8664,8666, 8668, 8670, 8672, 8674, 8676, 8678, 8680, 8682, 8684, 8686, 8688,8690, 8692, 8694, 8696, 8698, 8700, 8702, 8704, 8706, 8708, 8710, 8712,8714, 8716, 8718, 8720, 8722, 8724, 8726, 8728, 8730, 8732, 8734, 8736,8738, 8740, 8742, 8744, 8746, 8748, 8750, 8752, 8754, 8756, 8758, 8760,8762, 8764, 8766, 8768, 8770, 8772, 8774, 8776, 8778, 8780, 8782, 8784,8786, 8788, 8790, 8792, 8794, 8796, 9108, 9110, 9112, 9114, 9116, 9118,9120, 9122, 9124, 9126, 9128, 9130, 9132, 9134, 9136, 9138, 9140, 9142,9144, 9146, 9148, 9150, 9152, 9154, 9156, 9158, 9160, 9162, 9164, 9166,9168, 9170, 9172, 9174, 9176, 9178, 9180, 9182, 9184, 9186, 9188, 9190,9192, 9194, 9196, 9198, 9200, 9202, 9204, 9206, 9208, 9210, 9212, 9214,9216, 9218, 9220, and 9222. In some embodiments, the polypeptidesequence of the engineered glycosyltransferase comprises a sequence atleast 95% identical to any of SEQ ID NOS: 8624, 8626, 8628, 8630, 8632,8634, 8636, 8638, 8640, 8642, 8644, 8646, 8648, 8650, 8652, 8654, 8656,8658, 8660, 8662, 8664, 8666, 8668, 8670, 8672, 8674, 8676, 8678, 8680,8682, 8684, 8686, 8688, 8690, 8692, 8694, 8696, 8698, 8700, 8702, 8704,8706, 8708, 8710, 8712, 8714, 8716, 8718, 8720, 8722, 8724, 8726, 8728,8730, 8732, 8734, 8736, 8738, 8740, 8742, 8744, 8746, 8748, 8750, 8752,8754, 8756, 8758, 8760, 8762, 8764, 8766, 8768, 8770, 8772, 8774, 8776,8778, 8780, 8782, 8784, 8786, 8788, 8790, 8792, 8794, 8796, 9108, 9110,9112, 9114, 9116, 9118, 9120, 9122, 9124, 9126, 9128, 9130, 9132, 9134,9136, 9138, 9140, 9142, 9144, 9146, 9148, 9150, 9152, 9154, 9156, 9158,9160, 9162, 9164, 9166, 9168, 9170, 9172, 9174, 9176, 9178, 9180, 9182,9184, 9186, 9188, 9190, 9192, 9194, 9196, 9198, 9200, 9202, 9204, 9206,9208, 9210, 9212, 9214, 9216, 9218, 9220, and 9222. In some embodiments,the polypeptide sequence of the engineered glycosyltransferase comprisesany of SEQ ID NOS: 8624, 8626, 8628, 8630, 8632, 8634, 8636, 8638, 8640,8642, 8644, 8646, 8648, 8650, 8652, 8654, 8656, 8658, 8660, 8662, 8664,8666, 8668, 8670, 8672, 8674, 8676, 8678, 8680, 8682, 8684, 8686, 8688,8690, 8692, 8694, 8696, 8698, 8700, 8702, 8704, 8706, 8708, 8710, 8712,8714, 8716, 8718, 8720, 8722, 8724, 8726, 8728, 8730, 8732, 8734, 8736,8738, 8740, 8742, 8744, 8746, 8748, 8750, 8752, 8754, 8756, 8758, 8760,8762, 8764, 8766, 8768, 8770, 8772, 8774, 8776, 8778, 8780, 8782, 8784,8786, 8788, 8790, 8792, 8794, 8796, 9108, 9110, 9112, 9114, 9116, 9118,9120, 9122, 9124, 9126, 9128, 9130, 9132, 9134, 9136, 9138, 9140, 9142,9144, 9146, 9148, 9150, 9152, 9154, 9156, 9158, 9160, 9162, 9164, 9166,9168, 9170, 9172, 9174, 9176, 9178, 9180, 9182, 9184, 9186, 9188, 9190,9192, 9194, 9196, 9198, 9200, 9202, 9204, 9206, 9208, 9210, 9212, 9214,9216, 9218, 9220, and 9222.

The present invention also provides engineered sucrose synthasecomprising a polypeptide sequence that is at least 60%, 65%, 70%, 75%,80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99%, or more sequence identity to SEQ ID NO:72. In some embodimentsthe present invention provides engineered sucrose synthase comprising apolypeptide sequence that is at least 60%, 65%, 70%, 75%, 80%, 85%, 86%,87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or moresequence identity to SEQ ID NO: 74, 1080, 1158, 1222, 1392, 1456, 1582,1764, 1804, 1840, 2064, 2432, 2510, 7506, and/or 8420. In someembodiments the present invention provides engineered sucrose synthasecomprising a polypeptide sequence that is at least 90% or more sequenceidentity to SEQ ID NO: 74, 1080, 1158, 1222, 1392, 1456, 1582, 1764,1804, 1840, 2064, 2432, 2510, 7506, and/or 8420. In some embodiments thepresent invention provides engineered sucrose synthase comprising apolypeptide sequence that is at least 91% or more sequence identity toSEQ ID NO: 74, 1080, 1158, 1222, 1392, 1456, 1582, 1764, 1804, 1840,2064, 2432, 2510, 7506, and/or 8420. In some embodiments the presentinvention provides engineered sucrose synthase comprising a polypeptidesequence that is at least 92% or more sequence identity to SEQ ID NO:74, 1080, 1158, 1222, 1392, 1456, 1582, 1764, 1804, 1840, 2064, 2432,2510, 7506, and/or 8420. In some embodiments the present inventionprovides engineered sucrose synthase comprising a polypeptide sequencethat is at least 93% or more sequence identity to SEQ ID NO: 74, 1080,1158, 1222, 1392, 1456, 1582, 1764, 1804, 1840, 2064, 2432, 2510, 7506,and/or 8420. In some embodiments the present invention providesengineered sucrose synthase comprising a polypeptide sequence that is atleast 94% or more sequence identity to SEQ ID NO: 74, 1080, 1158, 1222,1392, 1456, 1582, 1764, 1804, 1840, 2064, 2432, 2510, 7506, and/or 8420.In some embodiments the present invention provides engineered sucrosesynthase comprising a polypeptide sequence that is at least 95% or moresequence identity to SEQ ID NO: 74, 1080, 1158, 1222, 1392, 1456, 1582,1764, 1804, 1840, 2064, 2432, 2510, 7506, and/or 8420. In someembodiments the present invention provides engineered sucrose synthasecomprising a polypeptide sequence that is at least 96% or more sequenceidentity to SEQ ID NO: 74, 1080, 1158, 1222, 1392, 1456, 1582, 1764,1804, 1840, 2064, 2432, 2510, 7506, and/or 8420. In some embodiments thepresent invention provides engineered sucrose synthase comprising apolypeptide sequence that is at least 97% or more sequence identity toSEQ ID NO: 74, 1080, 1158, 1222, 1392, 1456, 1582, 1764, 1804, 1840,2064, 2432, 2510, 7506, and/or 8420. In some embodiments the presentinvention provides engineered sucrose synthase comprising a polypeptidesequence that is at least 98% or more sequence identity to SEQ ID NO:74, 1080, 1158, 1222, 1392, 1456, 1582, 1764, 1804, 1840, 2064, 2432,2510, 7506, and/or 8420. In some embodiments the present inventionprovides engineered sucrose synthase comprising a polypeptide sequencethat is at least 99% or more sequence identity to SEQ ID NO: 74, 1080,1158, 1222, 1392, 1456, 1582, 1764, 1804, 1840, 2064, 2432, 2510, 7506,and/or 8420. In some embodiments the present invention providesengineered sucrose synthase comprising a polypeptide sequence of SEQ IDNO: 74, 1080, 1158, 1222, 1392, 1456, 1582, 1764, 1804, 1840, 2064,2432, 2510, 7506, and/or 8420. In additional embodiments, the presentinvention provides sucrose synthases provided in Table 18.1, 19.1, 19.2,20.1, 20.2, 20.3, 31.2, 31.3, 32.1, 32.2, 33.1, 33.2, 34.1, 34.2, 35.1,35.2, 36.1, 36.2, 37.1, 37.2, 37.3, 38.1, 38.2, 38.3, 39.1, 39.2, 39.3,40.1, 40.2, 40.3, 41.1, 41.2, 42.1, and/or 42.2. The present inventionalso provides engineered sucrose synthase comprising a polypeptidesequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99%, or more sequence identity to SEQ ID NO: 1080, 1082, 1084, 1086,1088, 1090, 1092, 1094, 1096, 1098, 1100, 1102, 1104, 1106, 1108, 1110,1112, 1114, 1116, 1118, 1120, 1122, 1124, 1126, 1128, 1130, 1132, 1134,1136, 1138, 1140, 1142, 1144, 1146, 1148, 1150, 1152, 1154, 1156, 1158,1160, 1162, 1164, 1166, 1168, 1170, 1172, 1174, 1176, 1178, 1180, 1182,1184, 1186, 1188, 1190, 1192, 1194, 1196, 1198, 1200, 1202, 1204, 1206,1208, 1210, 1212, 1214, 1216, 1218, 1220, 1222, 1224, 1226, 1228, 1230,1232, 1234, 1236, 1238, 1240, 1242, 1244, 1246, 1248, 1250, 1252, 1254,1256, 1258, 1260, 1262, 1264, 1266, 1268, 1270, 1272, 1274, 1276, 1278,1280, 1282, 1284, 1286, 1288, 1296, 1298, 1300, 1302, 1304, 1306, 1308,1310, 1312, 1314, 1316, 1318, 1320, 1322, 1324, 1326, 1328, 1330, 1332,1334, 1336, 1338, 1340, 1342, 1344, 1346, 1348, 1350, 1352, 1354, 1356,1358, 1360, 1362, 1364, 1366, 1368, 1370, 1372, 1374, 1376, 1378, 1380,1382, 1384, 1386, 1388, 1390, 1392, 1394, 1396, 1398, 1400, 1402, 1404,1406, 1408, 1410, 1412, 1414, 1416, 1418, 1420, 1422, 1424, 1426, 1428,1430, 1432, 1434, 1436, 1438, 1440, 1442, 1444, 1446, 1448, 1450, 1452,1454, 1456, 1458, 1460, 1462, 1464, 1466, 1468, 1470, 1472, 1474, 1476,1478, 1480, 1482, 1484, 1486, 1488, 1490, 1492, 1494, 1496, 1498, 1500,1502, 1504, 1506, 1508, 1510, 1512, 1514, 1516, 1518, 1520, 1522, 1524,1526, 1528, 1530, 1532, 1534, 1536, 1538, 1540, 1542, 1544, 1546, 1548,1550, 1552, 1554, 1556, 1558, 1560, 1562, 1564, 1566, 1568, 1570, 1572,1574, 1576, 1578, 1580, 1582, 1584, 1586, 1588, 1590, 1592, 1594, 1596,1598, 1600, 1602, 1604, 1606, 1608, 1610, 1612, 1614, 1616, 1618, 1620,1622, 1624, 1626, 1628, 1630, 1632, 1634, 1636, 1638, 1640, 1642, 1644,1646, 1648, 1650, 1652, 1654, 1656, 1658, 1660, 1662, 1664, 1666, 1668,1670, 1672, 1674, 1676, 1678, 1680, 1682, 1684, 1686, 1688, 1690, 1692,1694, 1696, 1698, 1700, 1702, 1704, 1706, 1708, 1710, 1712, 1714, 1716,1718, 1720, 1722, 1724, 1726, 1728, 1730, 1732, 1734, 1736, 1738, 1740,1742, 1744, 1746, 1748, 1750, 1752, 1754, 1756, 1758, 1760, 1762, 1764,1766, 1768, 1770, 1772, 1774, 1776, 1778, 1780, 1782, 1784, 1786, 1788,1790, 1792, 1794, 1796, 1798, 1800, 1802, 1804, 1806, 1808, 1810, 1812,1814, 1816, 1818, 1820, 1822, 1824, 1826, 1828, 1830, 1832, 1834, 1836,1838, 1840, 1842, 1844, 1846, 1848, 1850, 1852, 1854, 1856, 1858, 1860,1862, 1864, 1866, 1868, 1870, 1872, 1874, 1876, 1878, 1880, 1882, 1884,1886, 1888, 1890, 1892, 1894, 1896, 1898, 1900, 1902, 1904, 1906, 1908,1910, 1912, 1914, 1916, 1918, 1920, 1922, 1924, 1926, 1928, 1930, 1932,1934, 1936, 1938, 1940, 1942, 1944, 1946, 1948, 1950, 1952, 1954, 1956,1958, 1960, 1962, 1964, 1966, 1968, 1970, 1972, 1974, 1976, 1978, 1980,1982, 1984, 1986, 1988, 1990, 1992, 1994, 1996, 1998, 2000, 2002, 2004,2006, 2008, 2010, 2012, 2014, 2016, 2018, 2020, 2022, 2024, 2026, 2028,2030, 2032, 2034, 2036, 2038, 2040, 2042, 2044, 2046, 2048, 2050, 2052,2054, 2056, 2058, 2060, 2062, 2064, 2066, 2068, 2070, 2072, 2074, 2076,2078, 2080, 2082, 2084, 2086, 2088, 2090, 2092, 2094, 2096, 2098, 2100,2102, 2104, 2106, 2108, 2110, 2112, 2114, 2116, 2118, 2120, 2122, 2124,2126, 2128, 2130, 2132, 2134, 2136, 2138, 2140, 2142, 2144, 2146, 2148,2150, 2152, 2154, 2156, 2158, 2160, 2162, 2164, 2166, 2168, 2170, 2172,2174, 2176, 2178, 2180, 2182, 2184, 2186, 2188, 2190, 2192, 2194, 2196,2198, 2200, 2202, 2204, 2206, 2208, 2210, 2212, 2214, 2216, 2218, 2220,2222, 2224, 2226, 2228, 2230, 2232, 2234, 2236, 2238, 2240, 2242, 2244,2246, 2248, 2250, 2252, 2254, 2256, 2258, 2260, 2262, 2264, 2266, 2268,2270, 2272, 2274, 2276, 2278, 2280, 2282, 2284, 2286, 2288, 2290, 2292,2294, 2296, 2298, 2300, 2302, 2304, 2306, 2308, 2310, 2312, 2314, 2316,2318, 2320, 2322, 2324, 2326, 2328, 2330, 2332, 2334, 2336, 2338, 2340,2342, 2344, 2346, 2348, 2350, 2352, 2354, 2356, 2358, 2360, 2362, 2364,2366, 2368, 2370, 2372, 2374, 2376, 2378, 2380, 2382, 2384, 2386, 2388,2390, 2392, 2394, 2396, 2398, 2400, 2402, 2404, 2406, 2408, 2410, 2412,2414, 2416, 2418, 2420, 2422, 2424, 2426, 2428, 2430, 2432, 2434, 2436,2438, 2440, 2442, 2444, 2446, 2448, 2450, 2452, 2454, 2456, 2458, 2460,2462, 2464, 2466, 2468, 2470, 2472, 2474, 2476, 2478, 2480, 2482, 2484,2486, 2488, 2490, 2492, 2494, 2496, 2498, 2500, 2502, 2504, 2506, 2508,2510, 2512, 2514, 2516, 2518, 2520, 2522, 2524, 2526, 2528, 2530, 2532,2534, 2536, 2538, 2540, 2542, 2544, 2546, 2548, 2550, 2552, 2554, 2556,2558, 2560, 2562, 2564, 2566, 2568, 2570, 2572, 2574, 2576, 2578, 2580,2582, 2584, 2586, 2588, 2590, 2592, 2594, 7438, 7440, 7442, 7444, 7446,7448, 7450, 7452, 7454, 7456, 7458, 7460, 7462, 7464, 7466, 7468, 7470,7472, 7474, 7476, 7478, 7480, 7482, 7484, 7486, 7488, 7490, 7492, 7494,7496, 7498, 7500, 7502, 7504, 7506, 7508, 7510, 7512, 7514, 7516, 7518,7520, 7522, 7524, 7526, 7528, 7530, 7532, 7534, 7536, 7538, 7540, 7542,7544, 7546, 7548, 7550, 7552, 7554, 7556, 7558, 7560, 7562, 7564, 7566,7568, 7570, 7572, 7574, 7576, 7578, 7580, 7582, 7584, 7586, 7588, 7590,7592, 7594, 7596, 7598, 7600, 7602, 7604, 7606, 7608, 7610, 7612, 7614,7616, 7618, 7620, 7622, 7624, 7626, 7628, 7630, 7632, 7634, 7636, 7638,7640, 7642, 7644, 7646, 7648, 7650, 7652, 7654, 7656, 7658, 7660, 7662,7664, 7666, 7668, 7670, 7672, 7674, 7676, 7678, 7680, 7682, 7684, 7686,7688, 7690, 7692, 7694, 7696, 7698, 7700, 7702, 7704, 7706, 7708, 7710,7712, 7714, 7716, 7718, 7720, 7722, 7724, 7726, 7728, 7730, 7732, 7734,7736, 7738, 7740, 7742, 7744, 7746, 7748, 7750, 7752, 7754, 7756, 7758,7760, 7762, 7764, 8370, 8372, 8374, 8376, 8378, 8380, 8382, 8384, 8386,8388, 8390, 8392, 8394, 8396, 8398, 8400, 8402, 8404, 8406, 8408, 8410,8412, 8414, 8416, 8418, 8420, 8422, 8424, 8426, 8428, 8430, 8432, 8434,8436, 8438, 8440, 8442, 8444, 8446, 8448, 8450, 8452, 8454, 8456, 8458,8460, 8462, 8464, 8466, 8468, 8470, 8472, 8474, 8476, 8478, 8480, 8798,8800, 8802, 8804, 8806, 8808, 8810, 8812, 8814, 8816, 8818, 8820, 8822,8824, 8826, 8828, 8830, 8832, 8834, 8836, 8838, 8840, 8842, 8844, 8846,8848, 8850, 8852, 8854, 8856, 8858, 8860, 8862, 8864, 8866, 8868, 8870,8872, 8874, 8876, 8878, 8880, 8882, 8884, 8886, 8888, 8890, 8892, 8894,8896, 8898, 8900, 8902, 8904, 8906, 8908, 8910, 8912, 8914, 8916, 8918,8920, 8922, 8924, 8926, 8928, 8930, 8932, 8934, 8936, 8938, 8940, 8942,8944, 8946, 8948, 8950, 8952, 8954, 8956, 8958, 8960, 8962, 8964, 8966,8968, 8970, 8972, 8974, 8976, 8978, 8980, 8982, 8984, 8986, 8988, 8990,8992, 8994, 8996, 8998, 9000, 9002, 9004, 9006, 9008, 9010, 9012, 9014,9016, 9018, 9020, 9022, 9024, 9026, 9028, 9030, 9032, 9034, 9036, 9038,9040, 9042, 9044, 9046, 9048, 9050, 9052, 9054, 9056, 9058, 9060, 9062,9064, 9066, 9068, 9070, 9072, 9074, 9076, 9078, 9080, 9082, 9084, 9086,9088, 9090, 9092, 9094, 9096, 9098, 9100, 9102, 9104, and/or 9106.

The present invention also provides engineered sucrose synthases,wherein the polypeptide sequence of the engineered sucrose synthasescomprise at least one mutation or mutation set at one or more positionsselected from 4/9/349/532, 4/13/113/343/532, 4/13/113/532,4/33/47/52/343/532, 4/47/52/532, 4/113/532, 4/13/113, 4/13/532,4/33/113, 4/343, 7, 8, 44, 95, 117/440, 136, 221, 343/532, 440, 444,478, 532, 583, 611, 615, 615/789, 695, 722, and 788, wherein thepositions are numbered with reference to SEQ ID NO:74. In someembodiments, the polypeptide sequence of the engineered sucrose synthasecomprises at least one mutation or mutation set at one or more positionsselected from 4E/9T/349H/532S, 4E/13R/113Q/343H/532S, 4E/13R/113Q/532S,4E/33Q/47H/52D/343H/532S, 4E/47H/52D/532S, 4E/113Q/532S, 4E/13R/113Q,4E/13R/532S, 4E/33Q/113Q, 4E/343H, 7T, 8M, 44K, 95S, 117D/440T, 136S,221A/H, 343S/532S, 440P/T, 444K/T, 478T/V, 532R/T, 583Q, 611V,615C/E/T/V, 615L/789D, 695L, 722Y, and 788E, wherein the positions arenumbered with reference to SEQ ID NO:74. In some embodiments, thepolypeptide sequence of the engineered sucrose synthase comprises atleast one mutation or mutation set at one or more positions selectedfrom A4E/L9T/Q349H/F532S, A4E/P13R/I113Q/V343H/F532S,A4E/P13R/I113Q/F532S, A4E/Y33Q/L47H/A52D/V343H/F532S,A4E/L47H/A52D/F532S, A4E/I113Q/F532S, A4E/P13R/I113Q, A4E/P13R/F532S,A4E/Y33Q/I113Q, A4E/V343H, Q7T, Q8M, R44K, Q95S, G117D/R440T, R136S,R221A/H, V343S/F532S, R440P/T, Q444K/T, R478T/V, F532R/T, R583Q, R611V,R615C/E/T/V, R615L/A789D, V695L, R722Y, and H788E, wherein the positionsare numbered with reference to SEQ ID NO:74. In some embodiments, thepolypeptide sequence of the engineered sucrose synthase comprises asequence at least 90% identical to any of SEQ ID NOS: 1080, 1082, 1084,1086, 1088, 1090, 1092, 1094, 1096, 1098, 1100, 1102, 1104, 1106, 1108,1110, 1112, 1114, 1116, 1118, 1120, 1122, 1124, 1126, 1128, 1130, 1132,1134, 1136, 1138, 1140, 1142, 1144, 1146, 1148, 1150, and/or 1152. Insome embodiments, the polypeptide sequence of the engineered sucrosesynthase comprises a sequence at least 95% identical to any of SEQ IDNOS: 1080, 1082, 1084, 1086, 1088, 1090, 1092, 1094, 1096, 1098, 1100,1102, 1104, 1106, 1108, 1110, 1112, 1114, 1116, 1118, 1120, 1122, 1124,1126, 1128, 1130, 1132, 1134, 1136, 1138, 1140, 1142, 1144, 1146, 1148,1150, and/or 1152. In some further embodiments, the polypeptide sequenceof the engineered sucrose synthase comprises any of SEQ ID NOS: 1080,1082, 1084, 1086, 1088, 1090, 1092, 1094, 1096, 1098, 1100, 1102, 1104,1106, 1108, 1110, 1112, 1114, 1116, 1118, 1120, 1122, 1124, 1126, 1128,1130, 1132, 1134, 1136, 1138, 1140, 1142, 1144, 1146, 1148, 1150, and/or1152.

The present invention also provides engineered sucrose synthases,wherein the polypeptide sequence of the engineered sucrose synthasescomprise at least one mutation or mutation set at one or more positionsselected from 8/221, 47/221, 68/129/248, 68/129/248/595/600/756,68/146/248/387/506/550, 68/189/272/316/477/719/756,75/105/154/215/264/345, 75/105/345/410/769, 75/105/530, 75/345/530,85/170/225/266/534, 87/125/230/267/375/464/708,93/129/506/550/595/719/756, 93/477/635, 95/136/788,95/201/478/583/724/788, 95/385/478/583/788, 95/440/478/724/788/792,95/444/478/603/792, 95/444/478/724/788, 95/478/724, 98/250,113/225/266/415, 126/314/499/549/589/755, 136/440/444/478/603,136/440/444/478/583/788, 136/444/478/583/788/792, 225/372/534, 266,306/358/703/776, 358/636/737, 440/444/583/724/788, 440/478, and 466,wherein the positions are numbered with reference to SEQ ID NO:1080. Insome embodiments, the polypeptide sequence of the engineered sucrosesynthase comprises at least one mutation or mutation set at one or morepositions selected from 8M/221H, 47L/221H, 68A/129E/248A,68A/129E/248A/595V/600I/756V, 68A/146N/248A/387I/506P/550H,68A/189R/272L/316I/477K/719C/756V, 75V/105E/154H/215F/264V/345T,75V/105E/345T/410S/769R, 75V/105E/530L, 75V/345T/530L,85V/170L/225E/266N/534H, 87E/125E/230D/267V/375Y/464F/708A,93V/129E/506P/550H/595V/719C/756V, 93V/477K/635S, 95S/136S/788E,95S/201E/478V/583Q/724H/788E, 95S/385L/478V/583Q/788E,95S/440T/478V/724H/788E/792S, 95S/444T/478V/603S/792S,95S/444T/478V/724H/788E, 95S/478T/724H, 98V/250D, 113L/225E/266N/415K,126L/314L/499H/549E/589E/755G, 136S/440P/444T/478T/603S,136S/440T/444T/478V/583Q/788E, 136S/444T/478V/583Q/788E/792S,225E/372V/534H, 266N, 306L/358E/703Y/776E, 358E/636Q/737I,440P/444T/583Q/724H/788E, 440T/478V, and 466, wherein the positions arenumbered with reference to SEQ ID NO:1080. In some embodiments, thepolypeptide sequence of the engineered sucrose synthase comprises atleast one mutation or mutation set at one or more positions selectedfrom Q8M/R221H, H47L/R221H, V68A/R129E/S248A,V68A/R129E/S248A/I595V/V600I/I756V, V68A/D146N/S248A/V387I/S506P/R550H,V68A/G189R/I272L/V316I/D477K/A719C/I756V,M75V/A105E/R154H/I215F/I264V/A345T, M75V/A105E/A345T/T410S/Q769R,M75V/A105E/P530L, M75V/A345T/P530L, R85V/I170L/A225E/R266N/E534H,I87E/T125E/N230D/I267V/W375Y/I464F/T708A,R93V/R129E/S506P/R550H/I595V/A719C/I756V, R93V/D477K/A635S,Q95S/R136S/H788E, Q95S/Q201E/R478V/R583Q/K724H/H788E,Q95S/R385L/R478V/R583Q/H788E, Q95S/R440T/R478V/K724H/H788E/M792S,Q95S/Q444T/R478V/G603S/M792S, Q95S/Q444T/R478V/K724H/H788E,Q95S/R478T/K724H, L98V/S250D, I113L/A225E/R266N/R415K,V126L/V314L/N499H/D549E/G589E/R755G, R136S/R440P/Q444T/R478T/G603S,R136S/R440T/Q444T/R478V/R583Q/H788E,R136S/Q444T/R478V/R583Q/H788E/M792S, A225E/I372V/E534H, R266N,R306L/P358E/N703Y/Q776E, P358E/L636Q/V737I,R440P/Q444T/R583Q/K724H/H788E, R440T/R478V, and V466I, wherein thepositions are numbered with reference to SEQ ID NO: 1080.

In some embodiments, the polypeptide sequence of the engineered sucrosesynthase comprises a sequence at least 90% identical to any of SEQ IDNOS: 1192, 1194, 1196, 1198, 1200, 1202, 1204, 1206, 1208, 1210, 1212,1214, 1216, 1218, and/or 1220. In some embodiments, the polypeptidesequence of the engineered sucrose synthase comprises a sequence atleast 95% identical to any of SEQ ID NOS: 1192, 1194, 1196, 1198, 1200,1202, 1204, 1206, 1208, 1210, 1212, 1214, 1216, 1218, and/or 1220. Insome embodiments, the polypeptide sequence of the engineered sucrosesynthase comprises any of SEQ ID NOS: 1192, 1194, 1196, 1198, 1200,1202, 1204, 1206, 1208, 1210, 1212, 1214, 1216, 1218, and/or 1220.

The present invention also provides engineered sucrose synthases,wherein the polypeptide sequence of the engineered sucrose synthasescomprise at least one mutation or mutation set at one or more positionsselected from 8/68/95/98/358/478/595/724/792,8/68/98/221/248/250/440/477/534/595/724, 8/68/788,8/93/95/98/136/221/595/600/788, 8/93/95/113/250/440/595/600/724/788,8/95/98/440/478/534/600/788, 8/136/248/478/788,47/75/85/105/125/129/170/635, 47/75/85/105/375/756/776,47/75/85/264/267/372/415/635, 47/75/85/87/129/375/776,47/75/85/87/170/372/756, 47/85/105/129/201/230/267/583,47/85/125/372/583/635/756, 47/85/170/756, 47/85/87/105/125/635,47/85/87/154/756, 47/125/129/375/756/776, 47/129/170/635,47/154/372/375/583/635/708/756, 68/93/95/358/440/444/478/534/595/603,68/93/95/444/788, 68/93/98/136/248/250/358/440/534/724,75/85/87/105/264/267/583/708, 75/85/129/154/264/375,85/125/215/375/415/635/776, 85/87/105/215/267/756,85/87/129/375/756/776, 87/125/129/170/230/756, 87/154/306/375/756,93/95/98/534/792, 95/440/444/724/788, 129/215/372/756, and 170/264/267,wherein the positions are numbered with reference to SEQ ID NO:1158. Insome embodiments, the polypeptide sequence of the engineered sucrosesynthase comprises at least one mutation or mutation set at one or morepositions selected from 8M/68A/95S/98V/358E/478T/595V/724H/792S,8M/68A/98V/221H/248A/250D/440P/477K/534H/595V/724H, 8M/68A/788E,8M/93V/95S/98V/136S/221H/595V/600I/788E,8M/93V/95S/113I/250D/440T/595V/600I/724H/788E,8M/95S/98V/440P/478V/534H/600I/788E, 8M/136S/248A/478V/788E,47L/75V/85V/105E/125E/129E/170L/635S, 47L/75V/85V/105E/375Y/756V/776E,47L/75V/85V/264V/267V/372V/415R/635S,H47L/M75V/R85V/I87E/R129E/W375Y/Q776E, 47L/75V/85V/87E/170L/372V/756V,47L/85V/105E/129E/201E/230D/267V/583Q, 47L/85V/125E/372V/583Q/635S/756V,47L/85V/170L/756V, 47L/85V/87E/105E/125E/635S, 47L/85V/87E/154H/756V,47L/125E/129E/375Y/756V/776E, 47L/129E/170L/635S,47L/154H/372V/375Y/583Q/635S/708A/756V,68A/93V/95S/358E/440T/444T/478V/534H/595V/603S, 68A/93V/95S/444T/788E,68A/93V/98V/136S/248A/250D/358E/440P/534H/724H,75V/85V/87E/105E/264V/267V/583Q/708A, 75V/85V/129E/154H/264V/375Y,85V/125E/215F/375Y/415R/635S/776E, 85V/87E/105E/215F/267V/756V,85V/87E/129E/375Y/756V/776E, 87E/125E/129E/170L/230D/756V,87E/154H/306L/375Y/756V, 93V/95S/98V/534H/792S, 95S/440P/444T/724H/788E,129E/215F/372V/756V, 170L/264V/267V,8M/68A/95S/98V/358E/478T/595V/724H/792S,8M/68A/98V/221H/248A/250D/440P/477K/534H/595V/724H, 8M/68A/788E,8M/93V/95S/98V/136S/221H/595V/600I/788E,8M/93V/95S/113I/250D/440T/595V/600I/724H/788E,8M/95S/98V/440P/478V/534H/600I/788E, 8M/136S/248A/478V/788E,47L/75V/85V/105E/125E/129E/170L/635S, 47L/75V/85V/105E/375Y/756V/776E,47L/75V/85V/264V/267V/372V/415R/635S, 47L/75V/85V/87E/129E/375Y/776E,47L/75V/85V/87E/170L/372V/756V, 47L/85V/105E/129E/201E/230D/267V/583Q,47L/85V/125E/372V/583Q/635S/756V, 47L/85V/170L/756V,47L/85V/87E/105E/125E/635S, 47L/85V/87E/154H/756V,47L/125E/129E/375Y/756V/776E, 47L/129E/170L/635S,47L/154H/372V/375Y/583Q/635S/708A/756V,68A/93V/95S/358E/440T/444T/478V/534H/595V/603S, 68A/93V/95S/444T/788E,68A/93V/98V/136S/248A/250D/358E/440P/534H/724H,75V/85V/87E/105E/264V/267V/583Q/708A, 75V/85V/129E/154H/264V/375Y,85V/125E/215F/375Y/415R/635S/776E, 85V/87E/105E/215F/267V/756V,85V/87E/129E/375Y/756V/776E, 87E/125E/129E/170L/230D/756V,87E/154H/306L/375Y/756V, 93V/95S/98V/534H/792S, 95S/440P/444T/724H/788E,129E/215F/372V/756V, and 170L/264V/267V, wherein the positions arenumbered with reference to SEQ ID NO:1158. In some further embodiments,the polypeptide sequence of the engineered sucrose synthase comprises atleast one mutation or mutation set at one or more positions selectedfrom Q8M/V68A/Q95S/L98V/P358E/R478T/I595V/K724H/M792S,Q8M/V68A/L98V/R221H/S248A/S250D/R440P/D477K/E534H/I595V/K724H,Q8M/V68A/H788E, Q8M/R93V/Q95S/L98V/R136S/R221H/I595V/V600I/H788E,Q8M/R93V/Q95S/L113I/S250D/R440T/I595V/V600I/K724H/H788E,Q8M/Q95S/L98V/R440P/R478V/E534H/V600I/H788E,Q8M/R136S/S248A/R478V/H788E,H47L/M75V/R85V/A105E/T125E/R129E/I170L/A635S,H47L/M75V/R85V/A105E/W375Y/I756V/Q776E,H47L/M75V/R85V/I264V/I267V/I372V/K415R/A635S,H47L/M75V/R85V/I87E/R129E/W375Y/Q776E,H47L/M75V/R85V/I87E/I170L/I372V/I756V,H47L/R85V/A105E/R129E/Q201E/N230D/I267V/R583Q,H47L/R85V/T125E/I372V/R583Q/A635S/I756V, H47L/R85V/I170L/I756V,H47L/R85V/I87E/A105E/T125E/A635S, H47L/R85V/I87E/R154H/I756V,H47L/T125E/R129E/W375Y/I756V/Q776E, H47L/R129E/I170L/A635S,H47L/R154H/I372V/W375Y/R583Q/A635S/T708A/I756V,V68A/R93V/Q95S/P358E/R440T/Q444T/R478V/E534H/I595V/G603S,V68A/R93V/Q95S/Q444T/H788E,V68A/R93V/L98V/R136S/S248A/S250D/P358E/R440P/E534H/K724H,M75V/R85V/I87E/A105E/I264V/I267V/R583Q/T708A,M75V/R85V/R129E/R154H/I264V/W375Y,R85V/T125E/I215F/W375Y/K415R/A635S/Q776E,R85V/I87E/A105E/I215F/I267V/I756V, R85V/I87E/R129E/W375Y/I756V/Q776E,I87E/T125E/R129E/I170L/N230D/I756V, I87E/R154H/R306L/W375Y/I756V,R93V/Q95S/L98V/E534H/M792S, Q95S/R440P/Q444T/K724H/H788E,R129E/I215F/I372V/I756V, and I170L/I264V/I267V, wherein the positionsare numbered with reference to SEQ ID NO: 1158. In some embodiments, thepolypeptide sequence of the engineered sucrose synthase comprises asequence at least 90% identical to any of SEQ ID NOS: 1222, 1224, 1226,1228, 1230, 1232, 1234, 1236, 1238, 1240, 1242, 1244, 1246, 1248, 1250,1252, 1254, 1256, 1258, 1260, 1262, 1264, 1266, 1268, 1270, 1272, 1274,1276, 1278, 1280, 1282, 1284, 1286, and/or 1288. In some furtherembodiments, the polypeptide sequence of the engineered sucrose synthasecomprises a sequence at least 95% identical to any of SEQ ID NOS: 1222,1224, 1226, 1228, 1230, 1232, 1234, 1236, 1238, 1240, 1242, 1244, 1246,1248, 1250, 1252, 1254, 1256, 1258, 1260, 1262, 1264, 1266, 1268, 1270,1272, 1274, 1276, 1278, 1280, 1282, 1284, 1286, and/or 1288. In someembodiments, the polypeptide sequence of the engineered sucrose synthasecomprises any of SEQ ID NOS: 1222, 1224, 1226, 1228, 1230, 1232, 1234,1236, 1238, 1240, 1242, 1244, 1246, 1248, 1250, 1252, 1254, 1256, 1258,1260, 1262, 1264, 1266, 1268, 1270, 1272, 1274, 1276, 1278, 1280, 1282,1284, 1286, and/or 1288.

The present invention also provides engineered sucrose synthases,wherein the polypeptide sequence of the engineered sucrose synthasescomprise at least one mutation or mutation set at one or more positionsselected from 47/68/93/98/358/440, 47/68/154/372/375,47/93/98/136/154/772/776, 47/93/98/154/372/375/776,47/93/98/358/583/635, 47/93/129/136/154/250/372/534/635/724,47/93/129/136/375/534/583, 47/93/358/372/375/440/724,47/93/358/372/375/776, 47/98/129/358/372/375/438/534,47/98/129/375/534/635/724/776, 47/98/372/375, 47/125/154,47/129/136/372/375/534, 47/129/248/250/372/375/534/724, 47/136/583/776,47/358/440/724, 47/358/635/776, 47/372/635/776,68/93/98/129/358/375/724, 68/93/154/358/372/440/776, 68/129/440,68/129/583/724, 68/136/724, 68/154/358/375, 68/154/534/635,68/375/440/534/724/776, 93/98/125/154/248, 93/98/125/154/250/440,93/98/129/154/248, 93/98/154/250/358/375/534, 93/98/154/635/776,93/98/534, 93/125/154/440/534, 93/129/250/358/372/375/583,93/154/248/724, 93/358/534/635, 98/129/375/583, 98/534/583/635,129/136/154/248/250/372/375/534, 129/136/375,129/154/248/250/358/375/534/635, 129/358/372/635, 154/250/358/375/583,154/358/375/534/776, 154/534/635/724, 372/375/776, 375/635, and 534/583,wherein the positions are numbered with reference to SEQ ID NO: 1222. Insome embodiments, the polypeptide sequence of the engineered sucrosesynthase comprises at least one mutation or mutation set at one or morepositions selected from 47L/68A/93V/98V/358E/440P,47L/68A/154H/372V/375Y, 47L/93V/98V/136S/154H/772G/776E,47L/93V/98V/154H/372V/375Y/776E, 47L/93V/98V/358E/583Q/635S,47L/93V/129E/1365/154H/250D/372V/534H/635S/724H,47L/93V/129E/136S/375Y/534H/583Q, 47L/93V/358E/372V/375Y/440P/724H,47L/93V/358E/372V/375Y/776E, 47L/98V/129E/358E/372V/375Y/438Q/534H,47L/98V/129E/375Y/534H/635S/724H/776E, 47L/98V/372V/375Y, 47L/125E/154H,47L/129E/136S/372V/375Y/534H, 47L/129E/248A/250D/372V/375Y/534H/724H,47L/136S/583Q/776E, 47L/358E/440P/724H, 47L/358E/635S/776E,47L/372V/635S/776E, 68A/93V/98V/129E/358E/375Y/724H,68A/93V/154H/358E/372V/440P/776E, 68A/129E/440P, 68A/129E/583Q/724H,68A/136S/724H, 68A/154H/358E/375Y, 68A/154H/534H/635S,68A/375Y/440P/534H/724H/776E, 93V/98V/125E/154H/248A,93V/98V/125E/154H/250D/440P, 93V/98V/129E/154H/248A,93V/98V/154H/250D/358E/375Y/534H, 93V/98V/154H/635S/776E, 93V/98V/534H,93V/125E/154H/440P/534H, 93V/129E/250D/358E/372V/375Y/583Q,93V/154H/248A/724H, 93V/358E/534H/635S, 98V/129E/375Y/583Q,98V/534H/583Q/635S, 129E/136S/154H/248A/250D/372V/375Y/534H,129E/136S/375Y, 129E/154H/248A/250D/358E/375Y/534H/635S,129E/358E/372V/635S, 154H/250D/358E/375Y/583Q, 154H/358E/375Y/534H/776E,154H/534H/635S/724H, 372V/375Y/776E, 375Y/635S, and 534H/583Q, whereinthe positions are numbered with reference to SEQ ID NO: 1222. In someembodiments, the polypeptide sequence of the engineered sucrose synthasecomprises at least one mutation or mutation set at one or more positionsselected from H47L/V68A/R93V/L98V/P358E/R440P,H47L/V68A/R154H/I372V/W375Y, H47L/R93V/L98V/R136S/R154H/E772G/Q776E,H47L/R93V/L98V/R154H/I372V/W375Y/Q776E,H47L/R93V/L98V/P358E/R583Q/A635S,H47L/R93V/R129E/R136S/R154H/S250D/I372V/E534H/A635S/K724H,H47L/R93V/R129E/R136S/W375Y/E534H/R583Q,H47L/R93V/P358E/I372V/W375Y/R440P/K724H,H47L/R93V/P358E/I372V/W375Y/Q776E,H47L/L98V/R129E/P358E/I372V/W375Y/H438Q/E534H,H47L/L98V/R129E/W375Y/E534H/A635S/K724H/Q776E, H47L/L98V/I372V/W375Y,H47L/T125E/R154H, H47L/R129E/R136S/I372V/W375Y/E534H,H47L/R129E/S248A/S250D/I372V/W375Y/E534H/K724H, H47L/R136S/R583Q/Q776E,H47L/P358E/R440P/K724H, H47L/P358E/A635S/Q776E, H47L/I372V/A635S/Q776E,V68A/R93V/L98V/R129E/P358E/W375Y/K724H,V68A/R93V/R154H/P358E/I372V/R440P/Q776E, V68A/R129E/R440P,V68A/R129E/R583Q/K724H, V68A/R136S/K724H, V68A/R154H/P358E/W375Y,V68A/R154H/E534H/A635S, V68A/W375Y/R440P/E534H/K724H/Q776E,R93V/L98V/T125E/R154H/S248A, R93V/L98V/T125E/R154H/S250D/R440P,R93V/L98V/R129E/R154H/S248A, R93V/L98V/R154H/S250D/P358E/W375Y/E534H,R93V/L98V/R154H/A635S/Q776E, R93V/L98V/E534H,R93V/T125E/R154H/R440P/E534H, R93V/R129E/S250D/P358E/I372V/W375Y/R583Q,R93V/R154H/S248A/K724H, R93V/P358E/E534H/A635S, L98V/R129E/W375Y/R583Q,L98V/E534H/R583Q/A635S, R129E/R136S/R154H/S248A/S250D/1372V/W375Y/E534H,R129E/R136S/W375Y, R129E/R154H/S248A/S250D/P358E/W375Y/E534H/A635S,R129E/P358E/1372V/A635S, R154H/S250D/P358E/W375Y/R583Q,R154H/P358E/W375Y/E534H/Q776E, R154H/E534H/A635S/K724H,I372V/W375Y/Q776E, W375Y/A635S, and E534H/R583Q, wherein the positionsare numbered with reference to SEQ ID NO: 1222. In some embodiments, thepolypeptide sequence of the engineered sucrose synthase comprises asequence at least 90% identical to any of SEQ ID NOS: 1296, 1298, 1300,1302, 1304, 1306, 1308, 1310, 1312, 1314, 1316, 1318, 1320, 1322, 1324,1326, 1328, 1330, 1332, 1334, 1336, 1338, 1340, 1342, 1344, 1346, 1348,1350, 1352, 1354, 1356, 1358, 1360, 1362, 1364, 1366, 1368, 1370, 1372,1374, 1376, 1378, 1380, 1382, 1384, 1386, 1388, 1390, and 1392. In someembodiments, the polypeptide sequence of the engineered sucrose synthasecomprises a sequence at least 95% identical to any of SEQ ID NOS: 1296,1298, 1300, 1302, 1304, 1306, 1308, 1310, 1312, 1314, 1316, 1318, 1320,1322, 1324, 1326, 1328, 1330, 1332, 1334, 1336, 1338, 1340, 1342, 1344,1346, 1348, 1350, 1352, 1354, 1356, 1358, 1360, 1362, 1364, 1366, 1368,1370, 1372, 1374, 1376, 1378, 1380, 1382, 1384, 1386, 1388, 1390, and1392. In some embodiments, the polypeptide sequence of the engineeredsucrose synthase comprises any of SEQ ID NOS: 1296, 1298, 1300, 1302,1304, 1306, 1308, 1310, 1312, 1314, 1316, 1318, 1320, 1322, 1324, 1326,1328, 1330, 1332, 1334, 1336, 1338, 1340, 1342, 1344, 1346, 1348, 1350,1352, 1354, 1356, 1358, 1360, 1362, 1364, 1366, 1368, 1370, 1372, 1374,1376, 1378, 1380, 1382, 1384, 1386, 1388, 1390, and 1392.

The present invention also provides engineered sucrose synthases,wherein the polypeptide sequence of the engineered sucrose synthasescomprise at least one mutation or mutation set at one or more positionsselected from 17/357/364/434/519/684, 17/357/434/519/684, 17/434/684,17/684, 54/97/118/307/694/727/738, 68/98/129/136, 68/98/129/136/154,68/98/129/136/154/534, 68/98/129/154/534, 68/98/129/154/635,68/98/136/154/534/635, 68/98/136/154/635, 68/98/154, 68/98/154/534,68/98/154/534/635, 68/129/136, 68/129/136/154, 68/129/136/154/464/635,68/129/136/534/635, 68/129/154, 68/129/154/765, 68/136/154/534/635,68/136/534/635, 68/136/635, 68/154, 68/154/534/635, 68/154/635,97/118/442/694/727/738, 98/129/136/154, 98/129/136/154/635,98/129/136/534, 98/129/136/635, 98/129/154, 98/129/154/534/635,98/129/534/635, 98/136/154/635, 98/136/534/635, 98/136/635, 98/154,98/154/534, 122, 129/136, 129/136/154/635, 129/136/534, 129/136/635,129/154/534, 129/154/635, 129/635, 132/136/154/534/635, 136/154/635,136/534/635, 136/603, 136/635, 154, 154/635, 157, 160, 161, 167, 253,285, 381, 519, 550, 563, 564, 635, and 785, wherein the positions arenumbered with reference to SEQ ID NO: 1392. In some embodiments, thepolypeptide sequence of the engineered sucrose synthase comprises atleast one mutation or mutation set at one or more positions selectedfrom 17D/357K/364R/434H/519T/684H, 17D/357K/434H/519T/684H,17D/434H/684H, 17D/684H, 54D/97V/118N/307E/694N/727E/738E,68A/98V/129E/136S, 68A/98V/129E/136S/154H, 68A/98V/129E/136S/154H/534H,68A/98V/129E/154H/534H, 68A/98V/129E/154H/635S,68A/98V/136S/154H/534H/635S, 68A/98V/136S/154H/635S, 68A/98V/154H,68A/98V/154H/534H, 68A/98V/154H/534H/635S, 68A/129E/136S,68A/129E/136S/154H, 68A/129E/136S/154H/464F/635S,68A/129E/136S/534H/635S, 68A/129E/154H, 68A/129E/154H/765H,68A/136S/154H/534H/635S, 68A/136S/534H/635S, 68A/1365/6355, 68A/154H,68A/154H/534H/635S, 68A/154H/635S, 97V/118N/442N/694N/727E/738E,98V/129E/136S/154H, 98V/129E/136S/154H/635S, 98V/129E/136S/534H,98V/129E/1365/6355, 98V/129E/154H, 98V/129E/154H/534H/635S,98V/129E/534H/635S, 98V/136S/154H/635S, 98V/136S/534H/635S,98V/136S/635S, 98V/154H, 98V/154H/534H, 122D, 122E, 129E/136S,129E/136S/154H/635S, 129E/136S/534H, 129E/1365/6355, 129E/154H/534H,129E/154H/635S, 129E/635S, 132C/136S/154H/534H/635S, 136S/154H/635S,136S/534H/635S, 136S/603D, 1365/6355, 154H, 154H/635S, 157A, 157F, 160A,160E, 160M, 160N, 1605, 160W, 161Q, 167E, 253G, 253T, 253V, 285A, 3815,519A, 519G, 519L, 5195, 519T, 5501, 550M, 550Q, 5505, 563V, 564A, 635D,635E, 635R, and 785D, wherein the positions are numbered with referenceto SEQ ID NO: 1392. In some embodiments, the polypeptide sequence of theengineered sucrose synthase comprises at least one mutation or mutationset at one or more positions selected fromY17D/Y357K/P364R/Y434H/F519T/F684H, Y17D/Y357K/Y434H/F519T/F684H,Y17D/Y434H/F684H, Y17D/F684H, G54D/A97V/A118N/N307E/G694N/L727E/A738E,V68A/L98V/R129E/R136S, V68A/L98V/R129E/R136S/R154H,V68A/L98V/R129E/R136S/R154H/E534H, V68A/L98V/R129E/R154H/E534H,V68A/L98V/R129E/R154H/A635S, V68A/L98V/R136S/R154H/E534H/A635S,V68A/L98V/R136S/R154H/A635S, V68A/L98V/R154H, V68A/L98V/R154H/E534H,V68A/L98V/R154H/E534H/A635S, V68A/R129E/R136S, V68A/R129E/R136S/R154H,V68A/R129E/R136S/R154H/I464F/A635S, V68A/R129E/R136S/E534H/A635S,V68A/R129E/R154H, V68A/R129E/R154H/D765H, V68A/R136S/R154H/E534H/A635S,V68A/R136S/E534H/A635S, V68A/R136S/A635S, V68A/R154H,V68A/R154H/E534H/A635S, V68A/R154H/A635S,A97V/A118N/H442N/G694N/L727E/A738E, L98V/R129E/R136S/R154H,L98V/R129E/R136S/R154H/A635S, L98V/R129E/R136S/E534H,L98V/R129E/R136S/A635S, L98V/R129E/R154H, L98V/R129E/R154H/E534H/A635S,L98V/R129E/E534H/A635S, L98V/R136S/R154H/A635S, L98V/R136S/E534H/A635S,L98V/R136S/A635S, L98V/R154H, L98V/R154H/E534H, A122D, A122E,R129E/R136S, R129E/R136S/R154H/A635S, R129E/R136S/E534H,R129E/R136S/A635S, R129E/R154H/E534H, R129E/R154H/A635S, R129E/A635S,R132C/R136S/R154H/E534H/A635S, R136S/R154H/A635S, R136S/E534H/A635S,R136S/G603D, R136S/A635S, R154H, R154H/A635S, G157A, G157F, F160A,F160E, F160M, F160N, F160S, F160W, S161Q, R167E, A253G, A253T, A253V,P285A, Q381S, F519A, F519G, F519L, F519S, F519T, R550I, R550M, R550Q,R550S, L563V, S564A, A635D, A635E, A635R, and P785D, wherein thepositions are numbered with reference to SEQ ID NO: 1392. In someembodiments, the polypeptide sequence of the engineered sucrose synthasecomprises a sequence at least 90% identical to any of SEQ ID NOS: 1394,1396, 1398, 1400, 1402, 1404, 1406, 1408, 1410, 1412, 1414, 1416, 1418,1420, 1422, 1424, 1426, 1428, 1430, 1432, 1434, 1436, 1438, 1440, 1442,1444, 1446, 1448, 1450, 1452, 1454, 1456, 1458, 1460, 1462, 1464, 1466,1468, 1470, 1472, 1474, 1476, 1478, 1480, 1482, 1484, 1486, 1488, 1490,1492, 1494, 1496, 1498, 1500, 1502, 1504, 1506, 1508, 1510, 1512, 1514,1516, 1518, 1520, 1522, 1524, 1526, 1528, 1530, 1532, 1534, 1536, 1538,1540, 1542, 1544, 1546, 1548, 1550, 1552, 1554, 1556, 1558, 1560, 1562,1564, and 1566. In some embodiments, the polypeptide sequence of theengineered sucrose synthase comprises a sequence at least 95% identicalto any of SEQ ID NOS: 1394, 1396, 1398, 1400, 1402, 1404, 1406, 1408,1410, 1412, 1414, 1416, 1418, 1420, 1422, 1424, 1426, 1428, 1430, 1432,1434, 1436, 1438, 1440, 1442, 1444, 1446, 1448, 1450, 1452, 1454, 1456,1458, 1460, 1462, 1464, 1466, 1468, 1470, 1472, 1474, 1476, 1478, 1480,1482, 1484, 1486, 1488, 1490, 1492, 1494, 1496, 1498, 1500, 1502, 1504,1506, 1508, 1510, 1512, 1514, 1516, 1518, 1520, 1522, 1524, 1526, 1528,1530, 1532, 1534, 1536, 1538, 1540, 1542, 1544, 1546, 1548, 1550, 1552,1554, 1556, 1558, 1560, 1562, 1564, and 1566. In some embodiments, thepolypeptide sequence of the engineered sucrose synthase comprises any ofSEQ ID NOS: 1394, 1396, 1398, 1400, 1402, 1404, 1406, 1408, 1410, 1412,1414, 1416, 1418, 1420, 1422, 1424, 1426, 1428, 1430, 1432, 1434, 1436,1438, 1440, 1442, 1444, 1446, 1448, 1450, 1452, 1454, 1456, 1458, 1460,1462, 1464, 1466, 1468, 1470, 1472, 1474, 1476, 1478, 1480, 1482, 1484,1486, 1488, 1490, 1492, 1494, 1496, 1498, 1500, 1502, 1504, 1506, 1508,1510, 1512, 1514, 1516, 1518, 1520, 1522, 1524, 1526, 1528, 1530, 1532,1534, 1536, 1538, 1540, 1542, 1544, 1546, 1548, 1550, 1552, 1554, 1556,1558, 1560, 1562, 1564, and 1566.

The present invention also provides engineered sucrose synthases,wherein the polypeptide sequence of the engineered sucrose synthasescomprise at least one mutation or mutation set at one or more positionsselected from 17/54/97/136/329/550/684/738, 17/54/97/329/524/684,17/54/161/519/727/738, 17/54/524/550/727, 17/161/434/524/766,17/434/524/684, 17/434/738, 17/442/524/550/684/721, 17/727, 17/738,54/97/161/434/442, 54/97/434/524/550/684/727, 54/136/442/550,54/434/524/738, 97/136/519/550/727/738, and 329/550/684/727/738, whereinthe positions are numbered with reference to SEQ ID NO: 1456. In someembodiments, the polypeptide sequence of the engineered sucrose synthasecomprises at least one mutation or mutation set at one or more positionsselected from 17D/54D/97V/136P/329Q/550I/684H/738E,17D/54D/97V/329Q/524T/684H, 17D/54D/161T/519T/727E/738E,17D/54D/524T/550I/727E, 17D/161T/434H/524T/766H, 17D/434H/524T/684H,17D/434H/738E, 17D/442N/524T/550I/684H/721K, 17D/727E, 17D/738E,54D/97V/161T/434H/442N, 54D/97V/434H/524T/550I/684H/727E,54D/136P/442N/550I, 54D/434H/524T/738E, 97V/136P/519T/550I/727E/738E,and 329Q/550I/684H/727E/738E, wherein the positions are numbered withreference to SEQ ID NO: 1456. In some embodiments, the polypeptidesequence of the engineered sucrose synthase comprises at least onemutation or mutation set at one or more positions selected fromY17D/G54D/A97V/R136P/E329Q/R550I/F684H/A738E,Y17D/G54D/A97V/E329Q/A524T/F684H, Y17D/G54D/S161T/F519T/L727E/A738E,Y17D/G54D/A524T/R550I/L727E, Y17D/S161T/Y434H/A524T/R766H,Y17D/Y434H/A524T/F684H, Y17D/Y434H/A738E,Y17D/H442N/A524T/R550I/F684H/E721K, Y17D/L727E, Y17D/A738E,G54D/A97V/S161T/Y434H/H442N, G54D/A97V/Y434H/A524T/R550I/F684H/L727E,G54D/R136P/H442N/R550I, G54D/Y434H/A524T/A738E,A97V/R136P/F519T/R550I/L727E/A738E, and E329Q/R550I/F684H/L727E/A738E,wherein the positions are numbered with reference to SEQ ID NO: 1456. Insome embodiments, the polypeptide sequence of the engineered sucrosesynthase comprises a sequence at least 90% identical to any of SEQ IDNOS: 1568, 1570, 1572, 1574, 1576, 1578, 1580, 1582, 1584, 1586, 1588,1590, 1592, 1594, 1596, and 1598. In some embodiments, the polypeptidesequence of the engineered sucrose synthase comprises a sequence atleast 95% identical to any of SEQ ID NOS: 1568, 1570, 1572, 1574, 1576,1578, 1580, 1582, 1584, 1586, 1588, 1590, 1592, 1594, 1596, and 1598. Insome embodiments, the polypeptide sequence of the engineered sucrosesynthase comprises any of SEQ ID NOS: 1568, 1570, 1572, 1574, 1576,1578, 1580, 1582, 1584, 1586, 1588, 1590, 1592, 1594, 1596, and 1598.

The present invention also provides engineered sucrose synthases,wherein the polypeptide sequence of the engineered sucrose synthasescomprise at least one mutation or mutation set at one or more positionsselected from 14/137/356/745, 14/570, 14/570/745, 26/117/365,26/164/165/213/586, 71/158/222/356, 71/222/236, 71/319/356/606,117/158/213/332/608, 117/164/707, 117/213/365/517, 117/311/332, 117/608,122, 122/160/161/167/550, 122/160/161/282/381/550,122/160/161/282/381/550/636, 122/160/161/282/550, 122/160/161/550/636,122/160/167/282/381/550/636, 122/160/282/381, 122/160/282/381/550,122/160/282/550, 122/160/381/550, 122/160/381/550/636, 122/160/550,122/160/550/636, 122/161/550, 122/167, 122/167/550, 122/282/381/550,122/282/550, 122/282/550/636, 122/381/706, 122/550, 137/319/570,157/253/519, 160/161, 160/161/282/381/550, 160/161/282/550,160/161/550/636/735, 160/167/282/381/636, 160/282, 160/282/381/550,160/282/550/636, 160/381/550/636/681, 161/282/550/636, 161/381/550,165/311, 167/282/636, 167/550, 213/365/517/707, 236, 253/519,253/519/563, 253/519/635, 253/563/635, 270/322/517,270/367/452/517/613/700/750, 270/452/517/700/750, 270/570, 282/381/550,282/550, 356/570, 381/550, 517, 517/562/750, 517/640, 519/563, 550,550/636, and 562, wherein the positions are numbered with reference toSEQ ID NO: 1582. In some embodiments, the polypeptide sequence of theengineered sucrose synthase comprises at least one mutation or mutationset at one or more positions selected from 14D/137K/356H/745L, 14D/570H,14D/570H/745L, 26E/117E/365E, 26E/164E/165E/213E/586E,71Q/158T/222L/356H, 71Q/222L/2365, 71Q/319K/356H/6065,117E/158E/213E/332E/608E, 117E/164E/707E, 117E/213E/365E/517E,117E/311E/332E, 117E/608E, 122D, 122D/160M/282M/550S,122D/160W/161Q/167E/550M, 122D/160W/161Q/282M/550Q,122D/160W/282M/381S/550M, 122D/160W/550M, 122D/160W/550Q/636Q,122D/167E, 122D/282M/381S/550M, 122D/282M/550Q, 122D/550M,122E/160I/167E/282M/381H/550Q/636Q, 122E/160M/161Q/282M/381S/550M/636Q,122E/160M/550Q/636Q, 122E/160W/161Q/167E/550S,122E/160W/161Q/282M/3815/5505, 122E/160W/161Q/550M/636Q,122E/160W/282M/381s, 122E/160W/381S/550M/636Q, 122E/160W/381S/550Q,122E/161Q/550Q, 122E/167E/550M, 122E/282M/550M/636Q, 122E/282M/550Q,122E/282M/550S, 122E/3815/706K, 122E/550Q, 137K/319K/570H,157A/253T/519L, 160M/161Q, 160M/161Q/282M/381S/550M,160M/161Q/282M/550Q, 160M/282M, 160M/282M/381S/550M,160M/282M/550M/636Q, 160W/161Q/282M/550M, 160W/161Q/282M/550Q,160W/161Q/550Q/636Q/735V, 160W/167E/282M/381S/636Q, 160W/282M/381S/550M,160W/381S/550Q/636Q/681V, 161Q/282M/550Q/636Q, 161Q/381S/550Q,165E/311E, 167E/282M/636Q, 167E/550Q, 213E/365E/517E/707E, 2365,253G/519L, 253T/519L, 253T/519L/563V, 253T/519L/635D, 253T/519L/635E,253T/563V/635R, 253V/519G, 253V/519L, 270L/322V/517A,270L/367V/452Y/517A/613Q/700F/750M, 270L/452Y/517A/700F/750M, 270L/570H,282M/381S/550S, 282M/550Q, 356H/570H, 381S/550Q, 381S/550S, 517A,517A/562I/750M, 517A/640N, 519L/563V, 550M/636Q, 550Q, and 562, whereinthe positions are numbered with reference to SEQ ID NO: 1582. In someembodiments, the polypeptide sequence of the engineered sucrose synthasecomprises at least one mutation or mutation set at one or more positionsselected from R14D/R137K/R356H/R745L, R14D/R570H, R14D/R570H/R745L,S26E/G117E/H365E, S26E/A164E/A165E/A213E/R586E, R71Q/R158T/R222L/R356H,R71Q/R222L/R236S, R71Q/R319K/R356H/R606S, G117E/R158E/A213E/G332E/A608E,G117E/A164E/A707E, G117E/A213E/H365E/P517E, G117E/Q311E/G332E,G117E/A608E, A122D, A122D/F160M/L282M/R550S,A122D/F160W/T161Q/R167E/R550M, A122D/F160W/T161Q/L282M/R550Q,A122D/F160W/L282M/Q381S/R550M, A122D/F160W/R550M,A122D/F160W/R550Q/L636Q, A122D/R167E, A122D/L282M/Q381S/R550M,A122D/L282M/R550Q, A122D/R550M,A122E/F160I/R167E/L282M/Q381H/R550Q/L636Q,A122E/F160M/T161Q/L282M/Q381S/R550M/L636Q, A122E/F160M/R550Q/L636Q,A122E/F160W/T161Q/R167E/R550S, A122E/F160W/T161Q/L282M/Q381S/R550S,A122E/F160W/T161Q/R550M/L636Q, A122E/F160W/L282M/Q381S,A122E/F160W/Q381S/R550M/L636Q, A122E/F160W/Q381S/R550Q,A122E/T161Q/R550Q, A122E/R167E/R550M, A122E/L282M/R550M/L636Q,A122E/L282M/R550Q, A122E/L282M/R550S, A122E/Q381S/E706K, A122E/R550Q,R137K/R319K/R570H, G157A/A253T/T519L, F160M/T161Q,F160M/T161Q/L282M/Q381S/R550M, F160M/T161Q/L282M/R550Q, F160M/L282M,F160M/L282M/Q381S/R550M, F160M/L282M/R550M/L636Q,F160W/T161Q/L282M/R550M, F160W/T161Q/L282M/R550Q,F160W/T161Q/R550Q/L636Q/A735V, F160W/R167E/L282M/Q381S/L636Q,F160W/L282M/Q381S/R550M, F160W/Q381S/R550Q/L636Q/A681V,T161Q/L282M/R550Q/L636Q, T161Q/Q381S/R550Q, A165E/Q311E,R167E/L282M/L636Q, R167E/R550Q, A213E/H365E/P517E/A707E, R236S,A253G/T519L, A253T/T519L, A253T/T519L/L563V, A253T/T519L/S635D,A253T/T519L/S635E, A253T/L563V/S635R, A253V/T519G, A253V/T519L,V270L/I322V/P517A, V270L/I367V/F452Y/P517A/E613Q/I700F/L750M,V270L/F452Y/P517A/I700F/L750M, V270L/R570H, L282M/Q381S/R550S,L282M/R550Q, R356H/R570H, Q381S/R550Q, Q381S/R550S, P517A,P517A/L562I/L750M, P517A/T640N, T519L/L563V, R550M/L636Q, R550Q, andL562I, wherein the positions are numbered with reference to SEQ ID NO:1582. In some embodiments, the polypeptide sequence of the engineeredsucrose synthase comprises a sequence at least 90% identical to any ofSEQ ID NOS: 1600, 1602, 1604, 1606, 1608, 1610, 1612, 1614, 1616, 1618,1620, 1622, 1624, 1626, 1628, 1630, 1632, 1634, 1636, 1638, 1640, 1642,1644, 1646, 1648, 1650, 1652, 1654, 1656, 1658, 1660, 1662, 1664, 1666,1668, 1670, 1672, 1674, 1676, 1678, 1680, 1682, 1684, 1686, 1688, 1690,1692, 1694, 1696, 1698, 1700, 1702, 1704, 1706, 1708, 1710, 1712, 1714,1716, 1718, 1720, 1722, 1724, 1726, 1728, 1730, 1732, 1734, 1736, 1738,1740, 1742, 1744, 1746, 1748, 1750, 1752, 1754, 1756, 1758, 1760, 1762,1764, 1766, 1768, 1770, and 1772. In some embodiments, the polypeptidesequence of the engineered sucrose synthase comprises a sequence atleast 95% identical to any of SEQ ID NOS: 1600, 1602, 1604, 1606, 1608,1610, 1612, 1614, 1616, 1618, 1620, 1622, 1624, 1626, 1628, 1630, 1632,1634, 1636, 1638, 1640, 1642, 1644, 1646, 1648, 1650, 1652, 1654, 1656,1658, 1660, 1662, 1664, 1666, 1668, 1670, 1672, 1674, 1676, 1678, 1680,1682, 1684, 1686, 1688, 1690, 1692, 1694, 1696, 1698, 1700, 1702, 1704,1706, 1708, 1710, 1712, 1714, 1716, 1718, 1720, 1722, 1724, 1726, 1728,1730, 1732, 1734, 1736, 1738, 1740, 1742, 1744, 1746, 1748, 1750, 1752,1754, 1756, 1758, 1760, 1762, 1764, 1766, 1768, 1770, and 1772. In someembodiments, the polypeptide sequence of the engineered sucrose synthasecomprises any of SEQ ID NOS: 1600, 1602, 1604, 1606, 1608, 1610, 1612,1614, 1616, 1618, 1620, 1622, 1624, 1626, 1628, 1630, 1632, 1634, 1636,1638, 1640, 1642, 1644, 1646, 1648, 1650, 1652, 1654, 1656, 1658, 1660,1662, 1664, 1666, 1668, 1670, 1672, 1674, 1676, 1678, 1680, 1682, 1684,1686, 1688, 1690, 1692, 1694, 1696, 1698, 1700, 1702, 1704, 1706, 1708,1710, 1712, 1714, 1716, 1718, 1720, 1722, 1724, 1726, 1728, 1730, 1732,1734, 1736, 1738, 1740, 1742, 1744, 1746, 1748, 1750, 1752, 1754, 1756,1758, 1760, 1762, 1764, 1766, 1768, 1770, and 1772.

The present invention also provides engineered sucrose synthases,wherein the polypeptide sequence of the engineered sucrose synthasescomprise at least one mutation or mutation set at one or more positionsselected from 63/536, 117/122/270/540/681, 181/536/548, 181/536/548/705,181/548/705, 270/681, 347/532, 347/536/548/705, 407/570/681, 407/681,536, 536/548, 536/548/699, 536/705, 548, 548/580, 548/705, 580, 681,699, and 705, wherein the positions are numbered with reference to SEQID NO: 1764. In some embodiments, the polypeptide sequence of theengineered sucrose synthase comprises at least one mutation or mutationset at one or more positions selected from 63I/536L,117E/122D/270L/540M/681A, 181N/536L/548P, 181N/536L/548P/705M,181N/548P/705P, 270L/681A, 347R/532Y, 347R/536L/548P/705P,407I/570H/681A, 407T/681A, 536L, 536L/548P, 536L/548P/699F, 536L/705M,548P, 548P/580M, 548P/705P, 580M, 681A, 699F, 705M, and 705P, whereinthe positions are numbered with reference to SEQ ID NO: 1764. In someembodiments, the polypeptide sequence of the engineered sucrose synthasecomprises at least one mutation or mutation set at one or more positionsselected from A63I/E536L, G117E/A122D/V270L/L540M/V681A,G181N/E536L/A548P, G181N/E536L/A548P/H705M, G181N/A548P/H705P,V270L/V681A, N347R/S532Y, N347R/E536L/A548P/H705P, L407I/R570H/V681A,L407T/V681A, E536L, E536L/A548P, E536L/A548P/H699F, E536L/H705M, A548P,A548P/L580M, A548P/H705P, L580M, V681A, H699F, H705M, and H705P, whereinthe positions are numbered with reference to SEQ ID NO: 1764. In someembodiments, the polypeptide sequence of the engineered sucrose synthasecomprises a sequence at least 90% identical to any of SEQ ID NOS: 1774,1776, 1778, 1780, 1782, 1784, 1786, 1788, 1790, 1792, 1794, 1796, 1798,1800, 1802, 1804, 1806, 1808, 1810, 1812, 1814, 1816, 1818, and 1820. Insome embodiments, the polypeptide sequence of the engineered sucrosesynthase comprises a sequence at least 95% identical to any of SEQ IDNOS: 1774, 1776, 1778, 1780, 1782, 1784, 1786, 1788, 1790, 1792, 1794,1796, 1798, 1800, 1802, 1804, 1806, 1808, 1810, 1812, 1814, 1816, 1818,and 1820. In some embodiments, the polypeptide sequence of theengineered sucrose synthase comprises any of SEQ ID NOS: 1774, 1776,1778, 1780, 1782, 1784, 1786, 1788, 1790, 1792, 1794, 1796, 1798, 1800,1802, 1804, 1806, 1808, 1810, 1812, 1814, 1816, 1818, and 1820.

The present invention also provides engineered sucrose synthases,wherein the polypeptide sequence of the engineered sucrose synthasescomprise at least one mutation or mutation set at one or more positionsselected from 13, 17, 18, 30, 37, 52, 57, 60, 71, 85, 87, 90, 98, 99,118, 129, 164, 180, 183, 347/434/517/562/640/681,347/434/532/562/640/681, 347/434/550/562/681, 347/434/681,347/517/532/681, 347/532/550/640/681/699, 347/536/562/681,347/550/580/681, 347/550/681, 347/681, 365, 388, 389, 415, 433,434/517/532/681, 517/681, 531, 532/681, 535, 536/580/681, 539, 562/681,589, 606, 608, 707, 711, 727, 738, 748, 765, 769, and 789, wherein thepositions are numbered with reference to SEQ ID NO: 1804. In someembodiments, the polypeptide sequence of the engineered sucrose synthasecomprises at least one mutation or mutation set at one or more positionsselected from 13H, 13K, 13N, 17R, 18G, 30H, 37G, 37R, 52G, 52P, 52R,52W, 57R, 57W, 60L, 71G, 71Q, 85H, 87H, 87L, 90H, 98L, 991, 118G, 118N,129A, 129G, 129T, 164S, 164T, 180P, 183P, 347R/434H/517A/562I/640N/681A,347R/434H/532Y/562I/640N/681A, 347R/434H/550I/562I/681A, 347R/434H/681A,347R/517A/532Y/681A, 347R/532Y/550I/640N/681A/699F, 347R/536L/562I/681A,347R/550I/580M/681A, 347R/550I/681A, 347R/681A, 365W, 388K, 388R, 389G,415H, 433K, 433P, 434H/517A/532Y/681A, 517A/681A, 531A, 531R, 531T,532Y/681A, 535A, 535H, 535S, 536L/580M/681A, 539A, 539R, 562I/681A,589S, 606A, 606H, 606I, 606L, 606M, 606Q, 606V, 608P, 707G, 711K, 727K,738S, 748T, 765A, 765S, 769K, 769R, 789N, and 789R, wherein thepositions are numbered with reference to SEQ ID NO: 1804. In someembodiments, the polypeptide sequence of the engineered sucrose synthasecomprises at least one mutation or mutation set at one or more positionsselected from P13H, P13K, P13N, D17R, A18G, S30H, Q37G, Q37R, D52G,D52P, D52R, D52W, P57R, P57W, D60L, R71G, R71Q, V85H, E87H, E87L, R90H,V98L, E991, A118G, A118N, E129A, E129G, E129T, A164S, A164T, D180P,N183P, N347R/Y434H/P517A/L562I/T640N/V681A,N347R/Y434H/S532Y/L562I/T640N/V681A, N347R/Y434H/Q550I/L562I/V681A,N347R/Y434H/V681A, N347R/P517A/S532Y/V681A,N347R/S532Y/Q550I/T640N/V681A/H699F, N347R/E536L/L562I/V681A,N347R/Q550I/L580M/V681A, N347R/Q550I/V681A, N347R/V681A, H365W, L388K,L388R, A389G, K415H, L433K, L433P, Y434H/P517A/S532Y/V681A, P517A/V681A,S531A, S531R, S531T, S532Y/V681A, P535A, P535H, P535S,E536L/L580M/V681A, S539A, S539R, L562I/V681A, G589S, R606A, R606H,R606I, R606L, R606M, R606Q, R606V, A608P, A707G, R711K, E727K, E738S,E748T, D765A, D765S, Q769K, Q769R, A789N, and A789R, wherein thepositions are numbered with reference to SEQ ID NO: 1804. In someembodiments, the polypeptide sequence of the engineered sucrose synthasecomprises a sequence at least 90% identical to any of SEQ ID NOS: 1822,1824, 1826, 1828, 1830, 1832, 1834, 1836, 1838, 1840, 1842, 1844, 1846,1848, 1850, 1852, 1854, 1856, 1858, 1860, 1862, 1864, 1866, 1868, 1870,1872, 1874, 1876, 1878, 1880, 1882, 1884, 1886, 1888, 1890, 1892, 1894,1896, 1898, 1900, 1902, 1904, 1906, 1908, 1910, 1912, 1914, 1916, 1918,1920, 1922, 1924, 1926, 1928, 1930, 1932, 1934, 1936, 1938, 1940, 1942,1944, 1946, 1948, 1950, 1952, 1954, 1956, 1958, 1960, 1962, 1964, 1966,1968, 1970, 1972, 1974, 1976, 1978, 1980, 1982, and 1984. In someembodiments, the polypeptide sequence of the engineered sucrose synthasecomprises a sequence at least 95% identical to any of SEQ ID NOS: 1822,1824, 1826, 1828, 1830, 1832, 1834, 1836, 1838, 1840, 1842, 1844, 1846,1848, 1850, 1852, 1854, 1856, 1858, 1860, 1862, 1864, 1866, 1868, 1870,1872, 1874, 1876, 1878, 1880, 1882, 1884, 1886, 1888, 1890, 1892, 1894,1896, 1898, 1900, 1902, 1904, 1906, 1908, 1910, 1912, 1914, 1916, 1918,1920, 1922, 1924, 1926, 1928, 1930, 1932, 1934, 1936, 1938, 1940, 1942,1944, 1946, 1948, 1950, 1952, 1954, 1956, 1958, 1960, 1962, 1964, 1966,1968, 1970, 1972, 1974, 1976, 1978, 1980, 1982, and 1984. In someembodiments, the polypeptide sequence of the engineered sucrose synthasecomprises any of SEQ ID NOS: 1822, 1824, 1826, 1828, 1830, 1832, 1834,1836, 1838, 1840, 1842, 1844, 1846, 1848, 1850, 1852, 1854, 1856, 1858,1860, 1862, 1864, 1866, 1868, 1870, 1872, 1874, 1876, 1878, 1880, 1882,1884, 1886, 1888, 1890, 1892, 1894, 1896, 1898, 1900, 1902, 1904, 1906,1908, 1910, 1912, 1914, 1916, 1918, 1920, 1922, 1924, 1926, 1928, 1930,1932, 1934, 1936, 1938, 1940, 1942, 1944, 1946, 1948, 1950, 1952, 1954,1956, 1958, 1960, 1962, 1964, 1966, 1968, 1970, 1972, 1974, 1976, 1978,1980, 1982, and 1984.

The present invention also provides engineered sucrose synthases,wherein the polypeptide sequence of the engineered sucrose synthasescomprise at least one mutation or mutation set at one or more positionsselected from 17/52/87/118/129/388/589/738/765,17/52/87/118/129/589/738, 17/52/87/118/129/589/738/765,17/52/87/118/129/589/765, 17/52/87/129/388/589,17/52/87/129/388/589/738, 17/52/87/129/738, 17/52/87/388/589/765,17/52/87/589/738/765, 17/52/118/129/265/589/765,17/52/118/129/388/589/738/765, 17/52/118/129/589/738/765,17/52/118/129/738/765, 17/52/118/388/589/738, 17/52/118/388/589/738/765,17/52/118/388/738/765, 17/52/129/388/589/738, 17/52/129/388/589/738/765,17/52/129/589, 17/52/129/589/738, 17/52/129/589/765,17/52/129/653/738/765, 17/52/129/738, 17/52/129/738/765,17/52/388/589/738, 17/52/388/589/738/765, 17/52/589/738/765,17/52/589/765, 17/87/118/388/738, 17/87/129/388/738,17/118/129/388/738/765, 17/129/589, 17/129/589/738, 17/129/589/738/765,17/129/738/765, 17/388/589/738, 17/589/738, 17/589/765, 17/738/765,52/84/129/388/738/765, 52/87/118/129/388/765, 52/87/118/388/589/738,52/87/118/589/738/765, 52/87/129/388/738/765, 52/87/129/765,52/87/589/738, 52/87/738, 52/118/129/589/738, 52/118/129/765,52/118/388/589/738, 52/118/388/738/765, 52/129/589/738/765,52/129/589/765, 52/129/738, 52/388/738/765, 52/589/738, 52/589/738/765,52/738/765, 84/129/589/738/765, 87/118/129/765, 87/129/388/589, and589/738/765, wherein the positions are numbered with reference to SEQ IDNO: 1840. In some embodiments, the polypeptide sequence of theengineered sucrose synthase comprises at least one mutation or mutationset at one or more positions selected from17R/52G/87H/118N/129G/589S/738S, 17R/52G/87H/118N/129T/589S/765S,17R/52G/87H/129T/388K/589S/738S, 17R/52G/87H/129T/738S,17R/52G/87H/388K/589S/765S, 17R/52G/118N/129T/388K/589S/738S/765S,17R/52G/118N/388K/738S/765S, 17R/52G/129G/589S/738S,17R/52G/129T/388K/589S/738S, 17R/52G/129T/589S,17R/52G/129T/653H/738S/765S, 17R/52G/589S/765S,17R/52P/87H/118N/129T/388K/589S/738S/765S,17R/52P/87H/118N/129T/589S/738S/765S, 17R/52P/87H/129G/388K/589S,17R/52P/87H/129T/388K/589S/738S, 17R/52P/87H/589S/738S/765S,17R/52P/118N/129T/265T/589S/765S, 17R/52P/118N/129T/589S/738S/765S,17R/52P/118N/129T/738S/765S, 17R/52P/118N/388K/589S/738S,17R/52P/118N/388K/589S/738S/765S, 17R/52P/129G/388K/589S/738S/765S,17R/52P/129G/738S/765S, 17R/52P/129T/589S/765S, 17R/52P/129T/738S,17R/52P/388K/589S/738S, 17R/52P/388K/589S/738S/765S,17R/52P/589S/738S/765S, 17R/87H/118N/388K/738S, 17R/87H/129T/388K/738S,17R/118N/129T/388K/738S/765S, 17R/129T/589S, 17R/129T/589S/738S,17R/129T/589S/738S/765S, 17R/129T/738S/765S, 17R/388K/589S/738S,17R/589S/738S, 17R/589S/765S, 17R/738S/765S,52G/84A/129T/388K/738S/765S, 52G/87H/118N/589S/738S/765S,52G/87H/129T/388K/738S/765S, 52G/87H/738S, 52G/118N/388K/589S/738S,52G/118N/388K/738S/765S, 52G/129G/589S/765S, 52G/388K/738S/765S,52G/589S/738S/765S, 52G/738S/765S, 52P/87H/118N/129T/388K/765S,52P/87H/118N/388K/589S/738S, 52P/87H/129G/765S, 52P/87H/589S/738S,52P/118N/129T/589S/738S, 52P/118N/129T/765S, 52P/129T/589S/738S/765S,52P/129T/738S, 52P/589S/738S, 52P/589S/738S/765S,84A/129T/589S/738S/765S, 87H/118N/129T/765S, 87H/129T/388K/589S, and5895/7385/7655, wherein the positions are numbered with reference to SEQID NO: 1840. In some embodiments, the polypeptide sequence of theengineered sucrose synthase comprises at least one mutation or mutationset at one or more positions selected fromD17R/D52G/E87H/A118N/E129G/G589S/E738S,D17R/D52G/E87H/A118N/E129T/G589S/D765S,D17R/D52G/E87H/E129T/L388K/G589S/E738S, D17R/D52G/E87H/E129T/E738S,D17R/D52G/E87H/L388K/G589S/D765S,D17R/D52G/A118N/E129T/L388K/G589S/E738S/D765S,D17R/D52G/A118N/L388K/E738S/D765S, D17R/D52G/E129G/G589S/E738S,D17R/D52G/E129T/L388K/G589S/E738S, D17R/D52G/E129T/G589S,D17R/D52G/E129T/R653H/E738S/D765S, D17R/D52G/G589S/D765S,D17R/D52P/E87H/A118N/E129T/L388K/G589S/E738S/D765S,D17R/D52P/E87H/A118N/E129T/G589S/E738S/D765S,D17R/D52P/E87H/E129G/L388K/G589S,D17R/D52P/E87H/E129T/L388K/G589S/E738S,D17R/D52P/E87H/G589S/E738S/D765S,D17R/D52P/A118N/E129T/S265T/G589S/D765S,D17R/D52P/A118N/E129T/G589S/E738S/D765S,D17R/D52P/A118N/E129T/E738S/D765S, D17R/D52P/A118N/L388K/G589S/E738S,D17R/D52P/A118N/L388K/G589S/E738S/D765S,D17R/D52P/E129G/L388K/G589S/E738S/D765S, D17R/D52P/E129G/E738S/D765S,D17R/D52P/E129T/G589S/D765S, D17R/D52P/E129T/E738S,D17R/D52P/L388K/G589S/E738S, D17R/D52P/L388K/G589S/E738S/D765S,D17R/D52P/G589S/E738S/D765S, D17R/E87H/A118N/L388K/E738S,D17R/E87H/E129T/L388K/E738S, D17R/A118N/E129T/L388K/E738S/D765S,D17R/E129T/G589S, D17R/E129T/G589S/E738S, D17R/E129T/G589S/E738S/D765S,D17R/E129T/E738S/D765S, D17R/L388K/G589S/E738S, D17R/G589S/E738S,D17R/G589S/D765S, D17R/E738S/D765S, D52G/G84A/E129T/L388K/E738S/D765S,D52G/E87H/A118N/G589S/E738S/D765S, D52G/E87H/E129T/L388K/E738S/D765S,D52G/E87H/E738S, D52G/A118N/L388K/G589S/E738S,D52G/A118N/L388K/E738S/D765S, D52G/E129G/G589S/D765S,D52G/L388K/E738S/D765S, D52G/G589S/E738S/D765S, D52G/E738S/D765S,D52P/E87H/A118N/E129T/L388K/D765S, D52P/E87H/A118N/L388K/G589S/E738S,D52P/E87H/E129G/D765S, D52P/E87H/G589S/E738S,D52P/A118N/E129T/G589S/E738S, D52P/A118N/E129T/D765S,D52P/E129T/G589S/E738S/D765S, D52P/E129T/E738S, D52P/G589S/E738S,D52P/G589S/E738S/D765S, G84A/E129T/G589S/E738S/D765S,E87H/A118N/E129T/D765S, E87H/E129T/L388K/G589S, and G589S/E738S/D765S,wherein the positions are numbered with reference to SEQ ID NO: 1840. Insome embodiments, the polypeptide sequence of the engineered sucrosesynthase comprises at least one mutation or mutation set at one or morepositions selected from 14, 15, 18/362, 20, 24, 26, 33, 33/154, 46, 50,54, 58, 59, 59/72, 79, 81, 92, 93, 97/154, 104, 105, 130, 134, 154, 165,175, 185, 212, 213, 218, 241, 256, 263, 316, 319, 349, 360, 362, 364,390, 393, 434, 480, 498, 530, 534, 534/739, 542, 603, and 652, whereinthe positions are numbered with reference to SEQ ID NO: 1840. In someembodiments, the polypeptide sequence of the engineered sucrose synthasecomprises at least one mutation or mutation set at one or more positionsselected from 14K, 15A, 15I, 15P, 18V/362A, 20M, 245, 26A, 26E, 26I,26T, 33H/154C, 33L, 33P, 335, 46G, 46I, 46R, 46T, 46V, 50R, 54M, 58M,59A, 59C, 59N/72N, 59R, 595, 59V, 59W, 79H, 79Y, 81G, 81I, 81L, 92G,93T, 97V/154S, 104T, 105S, 130Y, 134A, 134P, 154A, 154E, 154R, 154S,1651, 165L, 165T, 175G, 175T, 185L, 212Y, 213V, 218A, 218N, 218Q, 218S,218T, 218V, 241T, 256G, 2635, 263Y, 316H, 316T, 319S, 349D, 349R, 349T,360D, 360E, 360R, 362E, 3645, 390M, 393H, 434G, 434R, 480P, 480V, 498L,498Q, 530F, 534G, 534K, 534L, 534R, 534T, 534W, 534W/739K, 542W, 603A,603E, 603H, 603Q, 603S, 652K, 652L, 652R, 652S, and 652T, wherein thepositions are numbered with reference to SEQ ID NO: 1840. In someembodiments, the polypeptide sequence of the engineered sucrose synthasecomprises at least one mutation or mutation set at one or more positionsselected from R14K, S15A, S15I, S15P, A18V/I362A, L20M, V24S, S26A,S26E, S26I, S26T, Q33H/H154C, Q33L, Q33P, Q33S, Q46G, Q46I, Q46R, Q46T,Q46V, G50R, D54M, L58M, E59A, E59C, E59N/D72N, E59R, E59S, E59V, E59W,W79H, W79Y, P81G, P81I, P81L, H92G, V93T, A97V/H154S, D104T, E105S,D130Y, V134A, V134P, H154A, H154E, H154R, H154S, A165I, A165L, A165T,S175G, S175T, M185L, W212Y, A213V, D218A, D218N, D218Q, D218S, D218T,D218V, L241T, S256G, M263S, M263Y, V316H, V316T, R319S, Q349D, Q349R,Q349T, G360D, G360E, G360R, I362E, P364S, E390M, S393H, Y434G, Y434R,I480P, I480V, E498L, E498Q, P530F, E534G, E534K, E534L, E534R, E534T,E534W, E534W/E739K, F542W, G603A, G603E, G603H, G603Q, G603S, G652K,G652L, G652R, G652S, and G652T, wherein the positions are numbered withreference to SEQ ID NO: 1840. In some embodiments, the polypeptidesequence of the engineered sucrose synthase comprises a sequence atleast 90% identical to any of SEQ ID NOS: 1986, 1988, 1990, 1992, 1994,1996, 1998, 2000, 2002, 2004, 2006, 2008, 2010, 2012, 2014, 2016, 2018,2020, 2022, 2024, 2026, 2028, 2030, 2032, 2034, 2036, 2038, 2040, 2042,2044, 2046, 2048, 2050, 2052, 2054, 2056, 2058, 2060, 2062, 2064, 2066,2068, 2070, 2072, 2074, 2076, 2078, 2080, 2082, 2084, 2086, 2088, 2090,2092, 2094, 2096, 2098, 2100, 2102, 2104, 2106, 2108, 2110, 2112, 2114,2116, 2118, 2120, 2122, 2124, 2126, 2128, 2130, 2132, 2134, 2136, 2138,2140, 2142, 2144, 2146, 2148, 2150, 2152, 2154, 2156, 2158, 2160, 2162,2164, 2166, 2168, 2170, 2172, 2174, 2176, 2178, 2180, 2182, 2184, 2186,2188, 2190, 2192, 2194, 2196, 2198, 2200, 2202, 2204, 2206, 2208, 2210,2212, 2214, 2216, 2218, 2220, 2222, 2224, 2226, 2228, 2230, 2232, 2234,2236, 2238, 2240, 2242, 2244, 2246, 2248, 2250, 2252, 2254, 2256, 2258,2260, 2262, 2264, 2266, 2268, 2270, 2272, 2274, 2276, 2278, 2280, 2282,2284, 2286, 2288, 2290, 2292, 2294, 2296, 2298, 2300, 2302, 2304, 2306,2308, 2310, 2312, 2314, 2316, and 2318. In some embodiments, thepolypeptide sequence of the engineered sucrose synthase comprises asequence at least 95% identical to any of SEQ ID NOS: 1986, 1988, 1990,1992, 1994, 1996, 1998, 2000, 2002, 2004, 2006, 2008, 2010, 2012, 2014,2016, 2018, 2020, 2022, 2024, 2026, 2028, 2030, 2032, 2034, 2036, 2038,2040, 2042, 2044, 2046, 2048, 2050, 2052, 2054, 2056, 2058, 2060, 2062,2064, 2066, 2068, 2070, 2072, 2074, 2076, 2078, 2080, 2082, 2084, 2086,2088, 2090, 2092, 2094, 2096, 2098, 2100, 2102, 2104, 2106, 2108, 2110,2112, 2114, 2116, 2118, 2120, 2122, 2124, 2126, 2128, 2130, 2132, 2134,2136, 2138, 2140, 2142, 2144, 2146, 2148, 2150, 2152, 2154, 2156, 2158,2160, 2162, 2164, 2166, 2168, 2170, 2172, 2174, 2176, 2178, 2180, 2182,2184, 2186, 2188, 2190, 2192, 2194, 2196, 2198, 2200, 2202, 2204, 2206,2208, 2210, 2212, 2214, 2216, 2218, 2220, 2222, 2224, 2226, 2228, 2230,2232, 2234, 2236, 2238, 2240, 2242, 2244, 2246, 2248, 2250, 2252, 2254,2256, 2258, 2260, 2262, 2264, 2266, 2268, 2270, 2272, 2274, 2276, 2278,2280, 2282, 2284, 2286, 2288, 2290, 2292, 2294, 2296, 2298, 2300, 2302,2304, 2306, 2308, 2310, 2312, 2314, 2316, and 2318. In some embodiments,the polypeptide sequence of the engineered sucrose synthase comprisesany of SEQ ID NOS: 1986, 1988, 1990, 1992, 1994, 1996, 1998, 2000, 2002,2004, 2006, 2008, 2010, 2012, 2014, 2016, 2018, 2020, 2022, 2024, 2026,2028, 2030, 2032, 2034, 2036, 2038, 2040, 2042, 2044, 2046, 2048, 2050,2052, 2054, 2056, 2058, 2060, 2062, 2064, 2066, 2068, 2070, 2072, 2074,2076, 2078, 2080, 2082, 2084, 2086, 2088, 2090, 2092, 2094, 2096, 2098,2100, 2102, 2104, 2106, 2108, 2110, 2112, 2114, 2116, 2118, 2120, 2122,2124, 2126, 2128, 2130, 2132, 2134, 2136, 2138, 2140, 2142, 2144, 2146,2148, 2150, 2152, 2154, 2156, 2158, 2160, 2162, 2164, 2166, 2168, 2170,2172, 2174, 2176, 2178, 2180, 2182, 2184, 2186, 2188, 2190, 2192, 2194,2196, 2198, 2200, 2202, 2204, 2206, 2208, 2210, 2212, 2214, 2216, 2218,2220, 2222, 2224, 2226, 2228, 2230, 2232, 2234, 2236, 2238, 2240, 2242,2244, 2246, 2248, 2250, 2252, 2254, 2256, 2258, 2260, 2262, 2264, 2266,2268, 2270, 2272, 2274, 2276, 2278, 2280, 2282, 2284, 2286, 2288, 2290,2292, 2294, 2296, 2298, 2300, 2302, 2304, 2306, 2308, 2310, 2312, 2314,2316, and 2318.

The present invention also provides engineered sucrose synthases,wherein the polypeptide sequence of the engineered sucrose synthasescomprise at least one mutation or mutation set at one or more positionsselected from 57/71/87/347/434/562/606, 57/71/129/180/434/536/562,57/71/129/434/531/536/562, 57/71/129/531/532/536/539/606,57/71/562/606/711/789, 57/71/789, 57/87/180/531/532/562/606/612/711,57/87/347/562, 57/90/129/562, 57/90/129/562/711,57/96/129/180/531/532/550/562, 57/129/347/531/532/539/562/711/747,57/129/347/536/550/562/711/789, 57/129/347/550/711,57/129/531/539/562/789, 57/129/536/606/789, 57/129/606, 57/180/562,57/180/562/606/612, 57/347/434/531/532/539/789,57/434/550/562/606/612/789, 57/531/532/536/562, 57/562/606/711,57/562/711, 71/129/180/347/531/539/550,71/129/180/434/532/536/539/711/789, 71/129/531, 71/129/606,71/347/532/550/562/711, 71/347/536/562/612/789, 71/536/539/562,87/189/532/536/562/711/789, 87/347/531/606/789, 87/347/536/539/550,90/129/539/550/606, 129/180/434/562/711/789, 129/180/606/711/789,129/347/562, 129/536/539/562, 129/539/562/789, 129/550, 129/550/562,129/562/606/711, 180/532, 180/550/606, 347/531/550/711,347/536/539/550/711/789, 347/536/562/606/612, 347/550/562/606,434/531/539/550/562/711, 434/550, 531/532/536/539/562/711/789,531/532/536/550/562/606/789, 531/532/562/606/711, 532/539/550, and562/711, wherein the positions are numbered with reference to SEQ ID NO:2064. In some embodiments, the polypeptide sequence of the engineeredsucrose synthase comprises at least one mutation or mutation set at oneor more positions selected from 57W/71Q/87H/347R/434H/562I/606H,57W/71Q/129G/531R/532Y/536L/539R/606H, 57W/71Q/129T/180P/434H/536L/562I,57W/71Q/129T/434H/531R/536L/562I, 57W/71Q/562I/606M/711K/789N,57W/71Q/789N, 57W/87H/180P/531R/532Y/562I/606M/612A/711K,57W/87H/347R/562I, 57W/90H/129G/562I, 57W/90H/129T/562I/711K,57W/96Q/129T/180P/531R/532Y/550I/562I, 57W/129G/531R/539R/562I/789N,57W/129G/536L/606M/789N, 57W/129T/347R/531R/532Y/539R/5621/711K/747V,57W/129T/347R/536L/5501/562I/711K/789N, 57W/129T/347R/5501/711K,57W/129T/606M, 57W/180P/562I, 57W/180P/5621/606H/612A,57W/347R/434H/531R/532Y/539R/789N, 57W/434H/550I/562I/606H/612A/789N,57W/531R/532Y/536L/562I, 57W/5621/606M/711K, 57W/5621/711K,71Q/129T/180P/347R/531R/539R/550I,71Q/129T/180P/434H/532Y/536L/539R/711K/789N, 71Q/129T/531R,71Q/129T/606M, 71Q/347R/532Y/550I/562I/711K,71Q/347R/536L/562I/612A/789N, 71Q/536L/539R/562I,87H/189D/532Y/536L/562I/711K/789N, 87H/347R/531R/606M/789N,87H/347R/536L/539R/550I, 90H/129T/539R/550I/606H, 129G/550I,129G/550I/562I, 129G/562I/606M/711K, 129T/180P/434H/562I/711K/789N,129T/180P/606M/711K/789N, 129T/347R/5621, 129T/536L/539R/5621,129T/539R/562/789N, 129T/550I/562I, 180P/532Y, 180P/550I/606M,347R/531R/550I/711K, 347R/536L/539R/5501/711K/789N,347R/536L/5621/606M/612A, 347R/5501/5621/606H,434H/531R/539R/550I/562I/711K, 434H/550I,531R/532Y/536L/539R/5621/711K/789N, 531R/532Y/536L/5501/5621/606M/789N,531R/532Y/562I/606H/711K, 532Y/539R/550I, and 5621/711K, wherein thepositions are numbered with reference to SEQ ID NO: 2064. In someembodiments, the polypeptide sequence of the engineered sucrose synthasecomprises at least one mutation or mutation set at one or more positionsselected from P57W/R71Q/E87H/N347R/Y434H/L562I/R606H,P57W/R71Q/E129G/S531R/S532Y/E536L/S539R/R606H,P57W/R71Q/E129T/D180P/Y434H/E536L/L562I,P57W/R71Q/E129T/Y434H/S531R/E536L/L562I,P57W/R71Q/L562I/R606M/R711K/A789N, P57W/R71Q/A789N,P57W/E87H/D180P/S531R/S532Y/L562I/R606M/E612A/R711K,P57W/E87H/N347R/L562I, P57W/R90H/E129G/L562I,P57W/R90H/E129T/L562I/R711K,P57W/L96Q/E129T/D180P/S531R/S532Y/Q550I/L562I,P57W/E129G/S531R/S539R/L562I/A789N, P57W/E129G/E536L/R606M/A789N,P57W/E129T/N347R/S531R/S532Y/S539R/L562I/R711K/A747V,P57W/E129T/N347R/E536L/Q550I/L562I/R711K/A789N,P57W/E129T/N347R/Q550I/R711K, P57W/E129T/R606M, P57W/D180P/L562I,P57W/D180P/L562I/R606H/E612A, P57W/N347R/Y434H/S531R/S532Y/S539R/A789N,P57W/Y434H/Q550I/L562I/R606H/E612A/A789N, P57W/S531R/S532Y/E536L/L562I,P57W/L562I/R606M/R711K, P57W/L562I/R711K,R71Q/E129T/D180P/N347R/S531R/S539R/Q550I,R71Q/E129T/D180P/Y434H/S532Y/E536L/S539R/R711K/A789N, R71Q/E129T/S531R,R71Q/E129T/R606M, R71Q/N347R/S532Y/Q550I/L562I/R711K,R71Q/N347R/E536L/L562I/E612A/A789N, R71Q/E536L/S539R/L562I,E87H/G189D/S532Y/E536L/L562I/R711K/A789N, E87H/N347R/S531R/R606M/A789N,E87H/N347R/E536L/S539R/Q550I, R90H/E129T/S539R/Q550I/R606H, E129G/Q550I,E129G/Q550I/L562I, E129G/L562I/R606M/R711K,E129T/D180P/Y434H/L562I/R711K/A789N, E129T/D180P/R606M/R711K/A789N,E129T/N347R/L562I, E129T/E536L/S539R/L562I, E129T/S539R/L562I/A789N,E129T/Q550I/L562I, D180P/S532Y, D180P/Q550I/R606M,N347R/S531R/Q550I/R711K, N347R/E536L/S539R/Q550I/R711K/A789N,N347R/E536L/L562I/R606M/E612A, N347R/Q550I/L562I/R606H,Y434H/S531R/S539R/Q550I/L562I/R711K, Y434H/Q550I,S531R/S532Y/E536L/S539R/L562I/R711K/A789N,S531R/S532Y/E536L/Q550I/L562I/R606M/A789N,S531R/S532Y/L562I/R606H/R711K, S532Y/S539R/Q550I, and L562I/R711K,wherein the positions are numbered with reference to SEQ ID NO: 2064. Insome embodiments, the polypeptide sequence of the engineered sucrosesynthase comprises at least one mutation or mutation set at one or morepositions selected from 21, 25/112, 41, 89, 91, 112, 186, 200, 226, 259,318, 330, 485, 487, 641, 674, 684, 688, 763, and 764, wherein thepositions are numbered with reference to SEQ ID NO: 2064.

In some embodiments, the polypeptide sequence of the engineered sucrosesynthase comprises at least one mutation or mutation set at one or morepositions selected from 21Q, 25T/112W, 41K, 89L, 89M, 91C, 91G, 112Q,112R, 186V, 200A, 226V, 259G, 318A, 330A, 485A, 4855, 4871, 487K, 487R,487T, 487V, 641L, 674A, 684G, 684H, 684M, 684T, 688A, 688F, 688G, 688H,688Q, 763L, and 764R, wherein the positions are numbered with referenceto SEQ ID NO: 2064. In some embodiments, the polypeptide sequence of theengineered sucrose synthase comprises at least one mutation or mutationset at one or more positions selected from R21Q, A25T/G112W, A41K, V89L,V89M, I91C, I91G, G112Q, G112R, L186V, V200A, P226V, D259G, P318A,S330A, G485A, G485S, Q487I, Q487K, Q487R, Q487T, Q487V, V641L, S674A,F684G, F684H, F684M, F684T, L688A, L688F, L688G, L688H, L688Q, V763L,and L764R, wherein the positions are numbered with reference to SEQ IDNO: 2064. In some embodiments, the polypeptide sequence of theengineered sucrose synthase comprises a sequence at least 90% identicalto any of SEQ ID NOS: 2320, 2322, 2324, 2326, 2328, 2330, 2332, 2334,2336, 2338, 2340, 2342, 2344, 2346, 2348, 2350, 2352, 2354, 2356, 2358,2360, 2362, 2364, 2366, 2368, 2370, 2372, 2374, 2376, 2378, 2380, 2382,2384, 2386, 2388, 2390, 2392, 2394, 2396, 2398, 2400, 2402, 2404, 2406,2408, 2410, 2412, 2414, 2416, 2418, 2420, 2422, 2424, 2426, 2428, 2430,2432, 2434, 2436, 2438, 2440, 2442, 2444, 2446, 2448, 2450, 2452, 2454,2456, 2458, 2460, 2462, 2464, 2466, 2468, 2470, 2472, 2474, 2476, 2478,2480, 2482, 2484, 2486, 2488, 2490, 2492, 2494, 2496, 2498, 2500, and2502. In some embodiments, the polypeptide sequence of the engineeredsucrose synthase comprises a sequence at least 95% identical to any ofSEQ ID NOS: 2320, 2322, 2324, 2326, 2328, 2330, 2332, 2334, 2336, 2338,2340, 2342, 2344, 2346, 2348, 2350, 2352, 2354, 2356, 2358, 2360, 2362,2364, 2366, 2368, 2370, 2372, 2374, 2376, 2378, 2380, 2382, 2384, 2386,2388, 2390, 2392, 2394, 2396, 2398, 2400, 2402, 2404, 2406, 2408, 2410,2412, 2414, 2416, 2418, 2420, 2422, 2424, 2426, 2428, 2430, 2432, 2434,2436, 2438, 2440, 2442, 2444, 2446, 2448, 2450, 2452, 2454, 2456, 2458,2460, 2462, 2464, 2466, 2468, 2470, 2472, 2474, 2476, 2478, 2480, 2482,2484, 2486, 2488, 2490, 2492, 2494, 2496, 2498, 2500, and 2502. In someembodiments, the polypeptide sequence of the engineered sucrose synthasecomprises any of SEQ ID NOS: 2320, 2322, 2324, 2326, 2328, 2330, 2332,2334, 2336, 2338, 2340, 2342, 2344, 2346, 2348, 2350, 2352, 2354, 2356,2358, 2360, 2362, 2364, 2366, 2368, 2370, 2372, 2374, 2376, 2378, 2380,2382, 2384, 2386, 2388, 2390, 2392, 2394, 2396, 2398, 2400, 2402, 2404,2406, 2408, 2410, 2412, 2414, 2416, 2418, 2420, 2422, 2424, 2426, 2428,2430, 2432, 2434, 2436, 2438, 2440, 2442, 2444, 2446, 2448, 2450, 2452,2454, 2456, 2458, 2460, 2462, 2464, 2466, 2468, 2470, 2472, 2474, 2476,2478, 2480, 2482, 2484, 2486, 2488, 2490, 2492, 2494, 2496, 2498, 2500,and 2502.

The present invention also provides engineered sucrose synthases,wherein the polypeptide sequence of the engineered sucrose synthasescomprise at least one mutation or mutation set at one or more positionsselected from 33/47/59/81/175/530/534/550/606,33/58/59/81/130/480/530/534/550/652, 33/58/59/480/530/534/550,33/58/154/480/534/550/603/606, 33/59/480/530/534/550/606,33/79/81/175/530/534, 33/79/81/175/530/534/603,33/79/154/480/530/534/550, 33/81/130/480/530/534/550,33/81/175/530/534/542/550/652, 33/130/530/534/550,33/154/480/530/534/603/606, 33/154/534, 33/530/534/550,58/59/79/175/480/534/550/652, 59/154/530/534/550,79/81/480/530/534/550/603/606/652, 81/480/530/534/550, and130/480/530/534/550/603/606, wherein the positions are numbered withreference to SEQ ID NO: 2432. In some embodiments, the polypeptidesequence of the engineered sucrose synthase comprises at least onemutation or mutation set at one or more positions selected from33H/47P/59A/81L/175G/530F/534W/550I/606M,33H/58M/59A/81G/130Y/480V/530F/534W/550I/652K,33H/58M/59A/480P/530F/534W/550I, 33H/58M/154A/480P/534W/550I/603A/606M,33H/59A/480P/530F/534W/550I/606M, 33H/79H/81G/175G/530F/534W,33H/79H/81L/175G/530F/534W/603Q, 33H/79H/154A/480P/530F/534W/550I,33H/81G/130Y/480V/530F/534W/550I, 33H/81I/175G/530F/534W/542W/550I/652R,33H/130Y/530F/534W/550I, 33H/154A/480P/530F/534W/603Q/606M,33H/154A/534W, 33H/530F/534W/550I, 58M/59A/79H/175G/480P/534W/550I/652R,59A/154A/530F/534W/550I, 79H/81I/480P/530F/534W/550I/603E/606M/652R,81G/480V/530F/534W/550I, and 130Y/480V/530F/534W/550I/603Q/606M, whereinthe positions are numbered with reference to SEQ ID NO: 2432. In someembodiments, the polypeptide sequence of the engineered sucrose synthasecomprises at least one mutation or mutation set at one or more positionsselected from Q33H/L47P/E59A/P81L/S175G/P530F/E534W/Q550I/R606M,Q33H/L58M/E59A/P81G/D130Y/I480V/P530F/E534W/Q550I/G652K,Q33H/L58M/E59A/I480P/P530F/E534W/Q550I,Q33H/L58M/H154A/I480P/E534W/Q550I/G603A/R606M,Q33H/E59A/I480P/P530F/E534W/Q550I/R606M,Q33H/W79H/P81G/S175G/P530F/E534W,Q33H/W79H/P81L/S175G/P530F/E534W/G603Q,Q33H/W79H/H154A/I480P/P530F/E534W/Q550I,Q33H/P81G/D130Y/I480V/P530F/E534W/Q550I,Q33H/P81I/S175G/P530F/E534W/F542W/Q550I/G652R,Q33H/D130Y/P530F/E534W/Q550I, Q33H/H154A/I480P/P530F/E534W/G603Q/R606M,Q33H/H154A/E534W, Q33H/P530F/E534W/Q550I,L58M/E59A/W79H/S175G/I480P/E534W/Q550I/G652R,E59A/H154A/P530F/E534W/Q550I,W79H/P81I/I480P/P530F/E534W/Q550I/G603E/R606M/G652R,P81G/I480V/P530F/E534W/Q550I, andD130Y/I480V/P530F/E534W/Q550I/G603Q/R606M, wherein the positions arenumbered with reference to SEQ ID NO: 2432. In some embodiments, thepolypeptide sequence of the engineered sucrose synthase comprises atleast one mutation or mutation set at one or more positions selectedfrom 25, 42, 70, 75, 77, 106, 199, 265, 267, 380, 410, 561, 642, and758, wherein the positions are numbered with reference to SEQ ID NO:2432. In some embodiments, the polypeptide sequence of the engineeredsucrose synthase comprises at least one mutation or mutation set at oneor more positions selected from 25E, 25G, 25L, 42H, 42S, 42T, 70H, 70N,70R, 70S, 70V, 75T, 75W, 77L, 77W, 106W, 199A, 265A, 265Q, 2671, 380T,410S, 561I, 561V, 642V, 758Q, and 758R, wherein the positions arenumbered with reference to SEQ ID NO: 2432. In some embodiments, thepolypeptide sequence of the engineered sucrose synthase comprises atleast one mutation or mutation set at one or more positions selectedfrom A25E, A25G, A25L, D42H, D42S, D42T, F70H, F70N, F70R, F70S, F70V,M75T, M75W, F77L, F77W, Y106W, T199A, S265A, S265Q, V267I, A380T, T410S,L561I, L561V, A642V, G758Q, and G758R, wherein the positions arenumbered with reference to SEQ ID NO: 2432. In some embodiments, thepolypeptide sequence of the engineered sucrose synthase comprises asequence at least 90% identical to any of SEQ ID NOS: 2504, 2506, 2508,2510, 2512, 2514, 2516, 2518, 2520, 2522, 2524, 2526, 2528, 2530, 2532,2534, 2536, 2538, 2540, 2542, 2544, 2546, 2548, 2550, 2552, 2554, 2556,2558, 2560, 2562, 2564, 2566, 2568, 2570, 2572, 2574, 2576, 2578, 2580,2582, 2584, 2586, 2588, 2590, 2592, and 2594. In some embodiments, thepolypeptide sequence of the engineered sucrose synthase comprises asequence at least 95% identical to any of SEQ ID NOS: 2504, 2506, 2508,2510, 2512, 2514, 2516, 2518, 2520, 2522, 2524, 2526, 2528, 2530, 2532,2534, 2536, 2538, 2540, 2542, 2544, 2546, 2548, 2550, 2552, 2554, 2556,2558, 2560, 2562, 2564, 2566, 2568, 2570, 2572, 2574, 2576, 2578, 2580,2582, 2584, 2586, 2588, 2590, 2592, and 2594. In some embodiments, thepolypeptide sequence of the engineered sucrose synthase comprises any ofSEQ ID NOS: 2504, 2506, 2508, 2510, 2512, 2514, 2516, 2518, 2520, 2522,2524, 2526, 2528, 2530, 2532, 2534, 2536, 2538, 2540, 2542, 2544, 2546,2548, 2550, 2552, 2554, 2556, 2558, 2560, 2562, 2564, 2566, 2568, 2570,2572, 2574, 2576, 2578, 2580, 2582, 2584, 2586, 2588, 2590, 2592, and2594.

The present invention also provides engineered sucrose synthases,wherein the polypeptide sequence of the engineered sucrose synthasescomprise at least one mutation or mutation set at one or more positionsselected from 41, 41/71, 41/71/112, 41/71/112/259/485/487/684/688,41/71/112/259/485/688, 41/71/259/485/532, 41/71/485, 41/71/485/532/684,41/71/487, 41/71/487/532/684, 41/71/532, 41/71/532/684, 41/71/684,41/84/259/485/487, 41/91/112/485, 41/91/112/485/487/532/684,41/91/112/485/532/684, 41/91/485, 41/112, 41/112/259/485/487,41/112/259/487/532/684, 41/112/485/684, 41/112/487/684, 41/112/532,41/112/684/688, 41/259/485, 41/259/485/487, 41/259/485/487/532/684,41/259/485/487/684/688, 41/259/532, 41/485, 41/485/487,41/485/487/684/688, 41/485/532, 41/485/532/688, 41/485/684/688,41/487/684, 41/532, 41/684, 41/684/688, 44/112/684/688,71/112/259/485/487/684, 71/112/485/688, 71/485/684/688, 71/532,71/684/688, 112, 112/259, 112/259/532/684/688, 112/259/684/688,112/485/684, 112/485/684/688, 226/487/684/688, 259/485/487/684,259/485/532, 259/487/684/688, 259/532, 485, 485/487, 485/487/532,485/487/532/684, 485/487/684, 485/487/684/688, 485/532, 485/684,485/684/688, 532, 532/684/688, 684, and 684/688, wherein the positionsare numbered with reference to SEQ ID NO: 2510. In some embodiments, thepolypeptide sequence of the engineered sucrose synthase comprises atleast one mutation or mutation set at one or more positions selectedfrom 41K, 41K/71Q, 41K/71Q/112Q, 41K/71Q/112Q/259G/485A/688Q,41K/71Q/112W/259G/485S/487R/684H/688Q, 41K/71Q/259G/485A/532Y,41K/71Q/485A, 41K/71Q/485A/532Y/684H, 41K/71Q/487R,41K/71Q/487R/532Y/684H, 41K/71Q/532Y, 41K/71Q/532Y/684H, 41K/71Q/684H,41K/84A/259G/485A/487I, 41K/91C/112Q/485S,41K/91C/112Q/485S/487K/532Y/684H, 41K/91C/112Q/485S/532Y/684H,41K/91C/485S, 41K/112Q/259G/485S/487R, 41K/112Q/259G/487R/532Y/684H,41K/112Q/485S/684H, 41K/112Q/487I/684H, 41K/112Q/532Y,41K/112Q/684H/688A, 41K/112W, 41K/259G/485A/487I,41K/259G/485A/487R/532Y/684H, 41K/259G/485A/487R/684H/688A,41K/259G/485S, 41K/259G/532Y, 41K/485A, 41K/485A/487K, 41K/485A/487R,41K/485A/532Y, 41K/485A/532Y/688G, 41K/485S/487I/684M/688G,41K/485S/684M/688A, 41K/487I/684M, 41K/487R/684M, 41K/532Y, 41K/684H,41K/684M/688A, 44C/112W/684H/688A, 71Q/112Q/259G/485A/487R/684M,71Q/112W/485S/688A, 71Q/485S/684M/688Q, 71Q/532Y, 71Q/684M/688A, 112Q,112Q/259G/532Y/684M/688G, 112Q/259G/684M/688Q, 112Q/485S/684H,112Q/485S/684H/688G, 112Q/485S/684M/688G, 112W, 112W/259G,226V/487I/684M/688A, 259G/485S/487K/684H, 259G/485S/532Y,259G/487I/684M/688G, 259G/532Y, 485A, 485A/487R, 485A/532Y,485A/684H/688G, 485A/684M, 4855, 485S/487I/684H/688Q, 485S/487I/684M,485S/487K/532Y, 485S/487R/532Y/684H, 485S/684M/688G, 532Y,532Y/684H/688Q, 684H, and 684M/688Q, wherein the positions are numberedwith reference to SEQ ID NO: 2510. In some embodiments, the polypeptidesequence of the engineered sucrose synthase comprises at least onemutation or mutation set at one or more positions selected from A41K,A41K/R71Q, A41K/R71Q/G112Q, A41K/R71Q/G112Q/D259G/G485A/L688Q,A41K/R71Q/G112W/D259G/G485S/Q487R/F684H/L688Q,A41K/R71Q/D259G/G485A/S532Y, A41K/R71Q/G485A,A41K/R71Q/G485A/S532Y/F684H, A41K/R71Q/Q487R,A41K/R71Q/Q487R/S532Y/F684H, A41K/R71Q/S532Y, A41K/R71Q/S532Y/F684H,A41K/R71Q/F684H, A41K/G84A/D259G/G485A/Q487I, A41K/I91C/G112Q/G485S,A41K/I91C/G112Q/G485S/Q487K/S532Y/F684H,A41K/I91C/G112Q/G485S/S532Y/F684H, A41K/I91C/G485S,A41K/G112Q/D259G/G485S/Q487R, A41K/G112Q/D259G/Q487R/S532Y/F684H,A41K/G112Q/G485S/F684H, A41K/G112Q/Q487I/F684H, A41K/G112Q/S532Y,A41K/G112Q/F684H/L688A, A41K/G112W, A41K/D259G/G485A/Q487I,A41K/D259G/G485A/Q487R/S532Y/F684H, A41K/D259G/G485A/Q487R/F684H/L688A,A41K/D259G/G485S, A41K/D259G/S532Y, A41K/G485A, A41K/G485A/Q487K,A41K/G485A/Q487R, A41K/G485A/S532Y, A41K/G485A/S532Y/L688G,A41K/G485S/Q487I/F684M/L688G, A41K/G485S/F684M/L688A, A41K/Q487I/F684M,A41K/Q487R/F684M, A41K/S532Y, A41K/F684H, A41K/F684M/L688A,R44C/G112W/F684H/L688A, R71Q/G112Q/D259G/G485A/Q487R/F684M,R71Q/G112W/G485S/L688A, R71Q/G485S/F684M/L688Q, R71Q/S532Y,R71Q/F684M/L688A, G112Q, G112Q/D259G/S532Y/F684M/L688G,G112Q/D259G/F684M/L688Q, G112Q/G485S/F684H, G112Q/G485S/F684H/L688G,G112Q/G485S/F684M/L688G, G112W, G112W/D259G, P226V/Q487I/F684M/L688A,D259G/G485S/Q487K/F684H, D259G/G485S/S532Y, D259G/Q487I/F684M/L688G,D259G/S532Y, G485A, G485A/Q487R, G485A/S532Y, G485A/F684H/L688G,G485A/F684M, G485S, G485S/Q487I/F684H/L688Q, G485S/Q487I/F684M,G485S/Q487K/S532Y, G485S/Q487R/S532Y/F684H, G485S/F684M/L688G, S532Y,S532Y/F684H/L688Q, F684H, and F684M/L688Q, wherein the positions arenumbered with reference to SEQ ID NO: 2510. In some embodiments, thepolypeptide sequence of the engineered sucrose synthase comprises atleast one mutation or mutation set at one or more positions selectedfrom 7/12, 12, 27, 29, 44, 45, 47, 48, 51, 55, 72, 95, 100, 116, 136,139, 176, 178, 198, 201, 205, 205/485, 207, 208, 280, 303, 317, 343,358, 361, 440, 478, 611, 615, 630, 675, 724, 756, and 788, wherein thepositions are numbered with reference to SEQ ID NO: 2510. In someembodiments, the polypeptide sequence of the engineered sucrose synthasecomprises at least one mutation or mutation set at one or more positionsselected from 7K/12L, 12C, 12N, 12Q, 12S, 12V, 27R, 29A, 29G, 29L, 29P,44L, 44V, 45A, 45C, 45G, 45L, 455, 45V, 47C, 47D, 47I, 47L, 47N, 47T,47V, 48A, 48V, 51P, 515, 55D, 55S, 72E, 95D, 95L, 95N, 95T, 95V, 100K,100P, 100Q, 116A, 116F, 136A, 136F, 136K, 136N, 136P, 136Q, 139K, 176M,176R, 176T, 176V, 178H, 198A, 198R, 2015, 205R/485S, 2055, 207K, 208K,280G, 303V, 3171, 343A, 343N, 358P, 358S, 361A, 361L, 361T, 440R, 478H,478I, 478Q, 611A, 615C, 615K, 615L, 615M, 630M, 675C, 724G, 724K, 724S,756C, and 788K, wherein the positions are numbered with reference to SEQID NO: 2510. In some embodiments, the polypeptide sequence of theengineered sucrose synthase comprises at least one mutation or mutationset at one or more positions selected from Q7K/D12L, D12C, D12N, D12Q,D12S, D12V, Q27R, D29A, D29G, D29L, D29P, R44L, R44V, E45A, E45C, E45G,E45L, E45S, E45V, P47C, P47D, P47I, P47L, P47N, P47T, P47V, P48A, P48V,Y51P, Y51S, I55D, I55S, D72E, Q95D, Q95L, Q95N, Q95T, Q95V, E100K,E100P, E100Q, L116A, L116F, R136A, R136F, R136K, R136N, R136P, R136Q,R139K, L176M, L176R, L176T, L176V, R178H, Q198A, Q198R, Q201S,T205R/G485S, T205S, P207K, R208K, K280G, R303V, E3171, H343A, H343N,E358P, E358S, R361A, R361L, R361T, P440R, R478H, R4781, R478Q, R611A,R615C, R615K, R615L, R615M, L630M, S675C, H724G, H724K, H724S, V756C,and H788K, wherein the positions are numbered with reference to SEQ IDNO: 2510. In some embodiments, the polypeptide sequence of theengineered sucrose synthase comprises a sequence at least 90% identicalto any of SEQ ID NOS: 7438, 7440, 7442, 7444, 7446, 7448, 7450, 7452,7454, 7456, 7458, 7460, 7462, 7464, 7466, 7468, 7470, 7472, 7474, 7476,7478, 7480, 7482, 7484, 7486, 7488, 7490, 7492, 7494, 7496, 7498, 7500,7502, 7504, 7506, 7508, 7510, 7512, 7514, 7516, 7518, 7520, 7522, 7524,7526, 7528, 7530, 7532, 7534, 7536, 7538, 7540, 7542, 7544, 7546, 7548,7550, 7552, 7554, 7556, 7558, 7560, 7562, 7564, 7566, 7568, 7570, 7572,7574, 7576, 7578, 7580, 7582, 7584, 7586, 7588, 7590, 7592, 7594, 7596,7598, 7600, 7602, 7604, 7606, 7608, 7610, 7612, 7614, 7616, 7618, 7620,7622, 7624, 7626, 7628, 7630, 7632, 7634, 7636, 7638, 7640, 7642, 7644,7646, 7648, 7650, 7652, 7654, 7656, 7658, 7660, 7662, 7664, 7666, 7668,7670, 7672, 7674, 7676, 7678, 7680, 7682, 7684, 7686, 7688, 7690, 7692,7694, 7696, 7698, 7700, 7702, 7704, 7706, 7708, 7710, 7712, 7714, 7716,7718, 7720, 7722, 7724, 7726, 7728, 7730, 7732, 7734, 7736, 7738, 7740,7742, 7744, 7746, 7748, 7750, 7752, 7754, 7756, 7758, 7760, 7762, and7764. In some embodiments, the polypeptide sequence of the engineeredsucrose synthase comprises a sequence at least 95% identical to any ofSEQ ID NOS: 7438, 7440, 7442, 7444, 7446, 7448, 7450, 7452, 7454, 7456,7458, 7460, 7462, 7464, 7466, 7468, 7470, 7472, 7474, 7476, 7478, 7480,7482, 7484, 7486, 7488, 7490, 7492, 7494, 7496, 7498, 7500, 7502, 7504,7506, 7508, 7510, 7512, 7514, 7516, 7518, 7520, 7522, 7524, 7526, 7528,7530, 7532, 7534, 7536, 7538, 7540, 7542, 7544, 7546, 7548, 7550, 7552,7554, 7556, 7558, 7560, 7562, 7564, 7566, 7568, 7570, 7572, 7574, 7576,7578, 7580, 7582, 7584, 7586, 7588, 7590, 7592, 7594, 7596, 7598, 7600,7602, 7604, 7606, 7608, 7610, 7612, 7614, 7616, 7618, 7620, 7622, 7624,7626, 7628, 7630, 7632, 7634, 7636, 7638, 7640, 7642, 7644, 7646, 7648,7650, 7652, 7654, 7656, 7658, 7660, 7662, 7664, 7666, 7668, 7670, 7672,7674, 7676, 7678, 7680, 7682, 7684, 7686, 7688, 7690, 7692, 7694, 7696,7698, 7700, 7702, 7704, 7706, 7708, 7710, 7712, 7714, 7716, 7718, 7720,7722, 7724, 7726, 7728, 7730, 7732, 7734, 7736, 7738, 7740, 7742, 7744,7746, 7748, 7750, 7752, 7754, 7756, 7758, 7760, 7762, and 7764. In someembodiments, the polypeptide sequence of the engineered sucrose synthasecomprises any of SEQ ID NOS: 7438, 7440, 7442, 7444, 7446, 7448, 7450,7452, 7454, 7456, 7458, 7460, 7462, 7464, 7466, 7468, 7470, 7472, 7474,7476, 7478, 7480, 7482, 7484, 7486, 7488, 7490, 7492, 7494, 7496, 7498,7500, 7502, 7504, 7506, 7508, 7510, 7512, 7514, 7516, 7518, 7520, 7522,7524, 7526, 7528, 7530, 7532, 7534, 7536, 7538, 7540, 7542, 7544, 7546,7548, 7550, 7552, 7554, 7556, 7558, 7560, 7562, 7564, 7566, 7568, 7570,7572, 7574, 7576, 7578, 7580, 7582, 7584, 7586, 7588, 7590, 7592, 7594,7596, 7598, 7600, 7602, 7604, 7606, 7608, 7610, 7612, 7614, 7616, 7618,7620, 7622, 7624, 7626, 7628, 7630, 7632, 7634, 7636, 7638, 7640, 7642,7644, 7646, 7648, 7650, 7652, 7654, 7656, 7658, 7660, 7662, 7664, 7666,7668, 7670, 7672, 7674, 7676, 7678, 7680, 7682, 7684, 7686, 7688, 7690,7692, 7694, 7696, 7698, 7700, 7702, 7704, 7706, 7708, 7710, 7712, 7714,7716, 7718, 7720, 7722, 7724, 7726, 7728, 7730, 7732, 7734, 7736, 7738,7740, 7742, 7744, 7746, 7748, 7750, 7752, 7754, 7756, 7758, 7760, 7762,and 7764.

The present invention also provides engineered sucrose synthases,wherein the polypeptide sequence of the engineered sucrose synthasescomprise at least one mutation or mutation set at one or more positionsselected from 8/25/55/95/208/358/440/517/788, 12/45/47,12/45/47/48/51/136/142/630, 12/45/47/51/136/139/630/758,12/45/47/136/139/142/675/758, 12/45/51/136/139/630/675/756/758,12/45/51/630/756, 12/48/51/136/139/758, 12/136/139/142/756/758,12/136/142, 12/630/756, 25/29/208/440, 25/100/154/208/440/517/705/788,25/517, 29/208/361/517/788, 42/198/199/480/532/539/561, 42/198/532/561,42/198/532/561/724, 42/199/480/532/561, 42/259/480/561, 42/259/480/652,42/480/561, 42/480/561/724, 42/561, 47/51/136/756/758, 55,55/410/440/603/788, 55/517, 55/517/788, 70, 70/642, 77/176/487/615/642,95/603, 106/199/539/561/652, 116, 136/139/142, 154/361/440/517/603/788,176, 198/199/480/561, 198/199/480/561/724, 198/199/561/724, 198/480/561,198/480/724, 199/532/539/561/652/724, 259/480, 267/611/642, 280/440/517,380, 480/561/652, 480/561/652/724/764, 480/724, 517, 532/539/561,532/561/724, 603, and 642, wherein the positions are numbered withreference to SEQ ID NO: 7506. In some embodiments, the polypeptidesequence of the engineered sucrose synthase comprises at least onemutation or mutation set at one or more positions selected from8R/25E/55D/95T/208K/358S/440V/517P/788K,12N/45A/47I/51P/136P/139K/630M/758R,12N/45A/51P/136P/139K/630M/675C/756C/758Q, 12N/48A/51P/136Q/139K/758Q,12N/136Q/142N, 12S/45A/51P/630M/756C, 12S/630M/756C,12V/45A/47I/48A/51S/136Q/142N/630M,12V/45A/47I/136Q/139K/142N/675C/758Q, 12V/45A/47L,12V/136Q/139K/142N/756C/758Q, 25E/29P/208K/440R,25E/100K/154A/208K/440R/517P/705M/788K, 25E/517P,29P/208K/361T/517P/788K, 42T/198R/199A/480V/532Y/539R/561V,42T/198R/532Y/561I/724K, 42T/198R/532Y/561V, 42T/199A/480V/532Y/561V,42T/259G/480V/561V, 42T/259G/480V/652K, 42T/480P/561I,42T/480V/561I/724K, 42T/561I, 47I/51S/136Q/756C/758Q, 55D,55D/410/440R/603E/788K, 55D/517P, 55D/517P/788K, 70N, 70N/642V,77L/176M/487K/615M/642V, 95T/603E, 106W/199A/539R/5611/652R, 116F,136P/139K/142N, 154A/361T/440R/517P/603E/788K, 176T,198R/199A/480P/561I, 198R/199A/480P/561V/724K, 198R/199A/561V/724G,198R/480P/561I, 198R/480V/724K, 199A/532Y/539R/561I/652R/7245,259G/480V, 2671/611Q/642V, 280G/440R/517P, 380T, 480V/561V/652R,480V/561V/652R/724K/764R, 480V/724K, 517P, 532Y/539R/561V,532Y/561I/724K, 603E, and 642V, wherein the positions are numbered withreference to SEQ ID NO: 7506. In some embodiments, the polypeptidesequence of the engineered sucrose synthase comprises at least onemutation or mutation set at one or more positions selected fromQ8R/A25E/I55D/Q95T/R208K/E358S/P440V/A517P/H788K,D12N/E45A/P47I/Y51P/R136P/R139K/L630M/G758R,D12N/E45A/Y51P/R136P/R139K/L630M/S675C/V756C/G758Q,D12N/P48A/Y51P/R136Q/R139K/G758Q, D12N/R136Q/S142N,D12S/E45A/Y51P/L630M/V756C, D12S/L630M/V756C,D12V/E45A/P47I/P48A/Y51S/R136Q/S142N/L630M,D12V/E45A/P47I/R136Q/R139K/S142N/S675C/G758Q, D12V/E45A/P47L,D12V/R136Q/R139K/S142N/V756C/G758Q, A25E/D29P/R208K/P440R,A25E/E100K/H154A/R208K/P440R/A517P/P705M/H788K, A25E/A517P,D29P/R208K/R361T/A517P/H788K, D42T/Q198R/T199A/I480V/S532Y/S539R/L561V,D42T/Q198R/S532Y/L561I/H724K, D42T/Q198R/S532Y/L561V,D42T/T199A/I480V/S532Y/L561V, D42T/D259G/I480V/L561V,D42T/D259G/I480V/G652K, D42T/I480P/L561I, D42T/I480V/L561I/H724K,D42T/L561I, P47I/Y51S/R136Q/V756C/G758Q, I55D,I55D/T4i0S/P440R/G603E/H788K, I55D/A517P, I55D/A517P/H788K, F70N,F70N/A642V, F77L/L176M/Q487K/R615M/A642V, Q95T/G603E,Y106W/T199A/S539R/L561I/G652R, L116F, R136P/R139K/S142N,H154A/R361T/P440R/A517P/G603E/H788K, L176T, Q198R/T199A/I480P/L561I,Q198R/T199A/I480P/L561V/H724K, Q198R/T199A/L561V/H724G,Q198R/I480P/L561I, Q198R/I480V/H724K,T199A/S532Y/S539R/L561I/G652R/H724S, D259G/I480V, V267I/R611Q/A642V,K280G/P440R/A517P, A380T, I480V/L561V/G652R,I480V/L561V/G652R/H724K/L764R, I480V/H724K, A517P, S532Y/S539R/L561V,S532Y/L561I/H724K, G603E, and A642V, wherein the positions are numberedwith reference to SEQ ID NO: 7506. In some embodiments, the polypeptidesequence of the engineered sucrose synthase comprises a sequence atleast 90% identical to any of SEQ ID NOS: 8370, 8372, 8374, 8376, 8378,8380, 8382, 8384, 8386, 8388, 8390, 8392, 8394, 8396, 8398, 8400, 8402,8404, 8406, 8408, 8410, 8412, 8414, 8416, 8418, 8420, 8422, 8424, 8426,8428, 8430, 8432, 8434, 8436, 8438, 8440, 8442, 8444, 8446, 8448, 8450,8452, 8454, 8456, 8458, 8460, 8462, 8464, 8466, 8468, 8470, 8472, 8474,8476, 8478, and 8480. In some embodiments, the polypeptide sequence ofthe engineered sucrose synthase comprises a sequence at least 95%identical to any of SEQ ID NOS: 8370, 8372, 8374, 8376, 8378, 8380,8382, 8384, 8386, 8388, 8390, 8392, 8394, 8396, 8398, 8400, 8402, 8404,8406, 8408, 8410, 8412, 8414, 8416, 8418, 8420, 8422, 8424, 8426, 8428,8430, 8432, 8434, 8436, 8438, 8440, 8442, 8444, 8446, 8448, 8450, 8452,8454, 8456, 8458, 8460, 8462, 8464, 8466, 8468, 8470, 8472, 8474, 8476,8478, and 8480. In some embodiments, the polypeptide sequence of theengineered sucrose synthase comprises any of SEQ ID NOS: 8370, 8372,8374, 8376, 8378, 8380, 8382, 8384, 8386, 8388, 8390, 8392, 8394, 8396,8398, 8400, 8402, 8404, 8406, 8408, 8410, 8412, 8414, 8416, 8418, 8420,8422, 8424, 8426, 8428, 8430, 8432, 8434, 8436, 8438, 8440, 8442, 8444,8446, 8448, 8450, 8452, 8454, 8456, 8458, 8460, 8462, 8464, 8466, 8468,8470, 8472, 8474, 8476, 8478, and 8480.

The present invention also provides engineered sucrose synthases,wherein the polypeptide sequence of the engineered sucrose synthasescomprise at least one mutation or mutation set at one or more positionsselected from 12/45/95/136/139/199/517/630/756, 12/45/95/136/756,12/45/136/139/199/517/603, 12/45/136/139/208/603/630/756,12/45/136/139/517/603/756, 12/45/136/139/517/630/642/756,12/45/136/139/517/756, 12/45/136/139/603/756, 12/45/136/139/642/756,12/45/136/176/517/603/630/642, 12/45/136/208/517/630/756,12/45/136/517/603/642/756/789, 12/45/136/517/630/642/756,12/45/136/603/756, 12/45/136/630/642, 12/45/139/176/208/517/603/630,12/45/139/199/208/603, 12/45/139/517/756, 12/45/139/756,12/45/176/603/630/642/756, 12/45/199/208/517/603/630/756,12/45/208/517/603/642/756, 12/95/136/139/517/603/756,12/95/139/517/630/756, 12/95/139/517/642, 12/95/139/630/642,12/95/199/517/642, 12/95/517/630/756, 12/95/630/756, 12/136,12/136/139/176/517/603/630, 12/136/139/176/517/603/756,12/136/139/176/630/756, 12/136/139/176/642, 12/136/139/176/756,12/136/139/199/208/517/603/756, 12/136/139/199/208/517/630/642/756,12/136/139/208/517, 12/136/139/517, 12/136/139/517/603/630/642/756,12/136/139/517/603/630/756, 12/136/139/517/603/756, 12/136/139/517/630,12/136/139/517/630/642, 12/136/139/517/630/642/756, 12/136/139/517/756,12/136/139/603/630, 12/136/139/603/630/642, 12/136/139/603/630/756,12/136/139/603/642/756, 12/136/139/630, 12/136/139/630/642/756,12/136/139/630/756, 12/136/139/642/756, 12/136/176/208/517/603/630/756,12/136/176/517/642, 12/136/176/603/756, 12/136/199/208/517/603/642/756,12/136/199/208/630/642, 12/136/199/517/756, 12/136/208,12/136/208/603/642, 12/136/517/603/630/642, 12/136/517/603/630/756,12/136/517/630, 12/136/517/630/642, 12/136/517/642, 12/136/517/642/756,12/136/517/756, 12/136/603/630/642/756, 12/136/603/630/756,12/136/603/642, 12/136/603/642/756, 12/136/603/756, 12/136/630,12/136/630/756, 12/139/176/517/603/630/756, 12/139/176/630/756,12/139/199/208/642, 12/139/199/517/630/756, 12/139/208/517,12/139/208/642, 12/139/517/603/642/756, 12/139/517/630/642/756,12/139/517/642, 12/139/603/642/756, 12/139/603/756, 12/139/630/642,12/139/630/642/756, 12/139/630/756, 12/176/517/603/630,12/176/517/630/642/756, 12/176/517/756, 12/176/603/630/756,12/176/603/756, 12/199/208/517/642, 12/199/630/642/756, 12/199/642/756,12/199/756, 12/208/517/603/623/630/642, 12/208/603/630/756,12/208/630/756, 12/517/603/630/642/756, 12/517/603/630/756,12/517/603/756, 12/517/630/642/756, 12/517/642/756, 12/603/630/756,12/603/642/756, 12/603/756, 12/630/642/756, 12/630/756,25/176/198/532/539, 116/142/198/434/440,136/139/176/199/208/517/630/642, 136/139/176/517/630/642/756,136/139/199/517/603/756, 136/139/208/517/630/756,136/139/208/603/630/756, 136/139/517/603/630/642/756,136/139/517/603/642/756, 136/139/517/603/756, 136/139/517/756,136/139/603, 136/139/630/642/756, 136/517/756, 136/603/756, 136/630/642,136/630/756, 136/642, 136/642/756, 136/756, 139/199/208/517/630/756,139/199/517/642, 139/208/517/630/642/756, 139/517/603/756,139/517/630/756, 139/642/756, 154/532/652/788, 199/517/603/630/756,208/517/630/642/756, 517/603/630/642/756, 517/630/756, 603/630/756, and603/756, wherein the positions are numbered with reference to SEQ ID NO:8420. In some embodiments, the polypeptide sequence of the engineeredsucrose synthase comprises at least one mutation or mutation set at oneor more positions selected from12N/45A/95T/136Q/139K/199A/517P/630M/756C,12N/45A/136Q/139K/199A/517P/603E, 12N/45A/136Q/139K/517P/603E/756C,12N/45A/136Q/139K/517P/630M/642V/756C, 12N/45A/136Q/139K/603E/756C,12N/45A/136Q/139K/642V/756C, 12N/45A/136Q/208K/517P/630M/756C,12N/45A/136Q/517P/630M/642V/756C, 12N/45A/136Q/603E/756C,12N/45A/136Q/630M/642V, 12N/45A/139K/199A/208K/603E,12N/45A/139K/517P/756C, 12N/45A/199A/208K/517P/603E/630M/756C,12N/95T/136Q/139K/517P/603Q/756C, 12N/95T/139K/517P/642V,12N/95T/139K/630M/642V, 12N/95T/199A/517P/642V, 12N/95T/517P/630M/756C,12N/136Q, 12N/136Q/139K/176T/517P/603Q/756C,12N/136Q/139K/176T/630M/756C, 12N/136Q/139K/176T/642V,12N/136Q/139K/199A/208K/517P/630M/642V/756C, 12N/136Q/139K/517P,12N/136Q/139K/517P/630M/642V, 12N/136Q/139K/517P/630M/642V/756C,12N/136Q/139K/517P/756C, 12N/136Q/139K/603E/630M,12N/136Q/139K/603Q/642V/756C, 12N/136Q/139K/630M,12N/136Q/139K/642V/756C, 12N/136Q/176T/603Q/756C,12N/136Q/199A/208K/517P/603E/642V/756C, 12N/136Q/199A/517P/756C,12N/136Q/208K, 12N/136Q/517P/630M, 12N/136Q/603E/642V,12N/136Q/603E/642V/756C, 12N/136Q/603Q/630M/756C,12N/136Q/603Q/642V/756C, 12N/136Q/603Q/756C, 12N/139K/199A/208K/642V,12N/139K/199A/517P/630M/756C, 12N/139K/208K/517P,12N/139K/517P/603Q/642V/756C, 12N/139K/603E/642V/756C,12N/139K/603Q/756C, 12N/139K/630M/642V, 12N/139K/630M/642V/756C,12N/139K/630M/756C, 12N/176T/517P/603E/630M,12N/176T/517P/630M/642V/756C, 12N/176T/517P/756C,12N/199A/208K/517P/642V, 12N/199A/630M/642V/756C, 12N/199A/642V/756C,12N/208K/517P/603E/623N/630M/642V, 12N/208K/630M/756C,12N/517P/603E/756C, 12N/517P/603Q/630M/756C, 12N/517P/630M/642V/756C,12N/603E/642V/756C, 12N/603Q/630M/756C, 12N/603Q/642V/756C,12N/603Q/756C, 12N/630M/642V/756C, 12S/45A/95T/136Q/756C,12S/45A/136Q/139K/208K/603E/630M/756C, 12S/45A/136Q/139K/517P/756C,12S/45A/136Q/176T/517P/603E/630M/642V,12S/45A/136Q/517P/603Q/642V/756C/789V,12S/45A/139K/176T/208K/517P/603E/630M, 12S/45A/139K/517P/756C,12S/45A/139K/756C, 12S/45A/176T/603Q/630M/642V/756C,12S/45A/208K/517P/603E/642V/756C, 12S/95T/139K/517P/630M/756C,12S/95T/630M/756C, 12S/136Q/139K/176T/517P/603Q/630M,12S/136Q/139K/176T/756C, 12S/136Q/139K/199A/208K/517P/603E/756C,12S/136Q/139K/208K/517P, 12S/136Q/139K/517P/603E/630M/642V/756C,12S/136Q/139K/517P/603E/630M/756C, 12S/136Q/139K/517P/603E/756C,12S/136Q/139K/517P/603Q/630M/642V/756C, 12S/136Q/139K/517P/603Q/756C,12S/136Q/139K/517P/630M, 12S/136Q/139K/517P/630M/642V,12S/136Q/139K/603Q/630M/642V, 12S/136Q/139K/603Q/630M/756C,12S/136Q/139K/630M/642V/756C, 12S/136Q/139K/630M/756C,12S/136Q/176T/208K/517P/603E/630M/756C, 12S/136Q/176T/517P/642V,12S/136Q/199A/208K/630M/642V, 12S/136Q/208K/603Q/642V,12S/136Q/517P/603Q/630M/642V, 12S/136Q/517P/603Q/630M/756C,12S/136Q/517P/630M/642V, 12S/136Q/517P/642V, 12S/136Q/517P/642V/756C,12S/136Q/517P/756C, 12S/136Q/603E/756C, 12S/136Q/603Q/630M/642V/756C,12S/136Q/630M, 12S/136Q/630M/756C, 12S/139K/176T/517P/603Q/630M/756C,12S/139K/176T/630M/756C, 12S/139K/208K/642V,12S/139K/517P/630M/642V/756C, 12S/139K/517P/642V, 12S/139K/603Q/756C,12S/176T/603E/756C, 12S/176T/603Q/630M/756C, 12S/199A/756C,12S/208K/603Q/630M/756C, 12S/517P/603Q/630M/642V/756C,12S/517P/630M/642V/756C, 12S/517P/642V/756C, 12S/603Q/630M/756C,12S/630M/756C, 25E/176M/198R/532Y/539R, 116F/142N/198R/434H/440R,136Q/139K/176T/199A/208K/517P/630M/642V,136Q/139K/176T/517P/630M/642V/756C, 136Q/139K/199A/517P/603E/756C,136Q/139K/208K/517P/630M/756C, 136Q/139K/208K/603Q/630M/756C,136Q/139K/517P/603E/642V/756C, 136Q/139K/517P/603Q/630M/642V/756C,136Q/139K/517P/603Q/756C, 136Q/139K/517P/756C, 136Q/139K/603Q,136Q/139K/630M/642V/756C, 136Q/517P/756C, 136Q/603Q/756C,136Q/630M/642V, 136Q/630M/756C, 136Q/642V, 136Q/642V/756C, 136Q/756C,139K/199A/208K/517P/630M/756C, 139K/199A/517P/642V,139K/208K/517P/630M/642V/756C, 139K/517P/603Q/756C, 139K/517P/630M/756C,139K/642V/756C, 154A/532Y/652R/788K, 199A/517P/603Q/630M/756C,208K/517P/630M/642V/756C, 517P/603E/630M/642V/756C, 517P/630M/756C,603E/630M/756C, and 603Q/756C, wherein the positions are numbered withreference to SEQ ID NO: 8420. In some embodiments, the polypeptidesequence of the engineered sucrose synthase comprises at least onemutation or mutation set at one or more positions selected fromD12N/E45A/Q95T/R136Q/R139K/T199A/A517P/L630M/V756C,D12N/E45A/R136Q/R139K/T199A/A517P/G603E,D12N/E45A/R136Q/R139K/A517P/G603E/V756C,D12N/E45A/R136Q/R139K/A517P/L630M/A642V/V756C,D12N/E45A/R136Q/R139K/G603E/V756C, D12N/E45A/R136Q/R139K/A642V/V756C,D12N/E45A/R136Q/R208K/A517P/L630M/V756C,D12N/E45A/R136Q/A517P/L630M/A642V/V756C, D12N/E45A/R136Q/G603E/V756C,D12N/E45A/R136Q/L630M/A642V, D12N/E45A/R139K/T199A/R208K/G603E,D12N/E45A/R139K/A517P/V756C,D12N/E45A/T199A/R208K/A517P/G603E/L630M/V756C,D12N/Q95T/R136Q/R139K/A517P/G603Q/V756C, D12N/Q95T/R139K/A517P/A642V,D12N/Q95T/R139K/L630M/A642V, D12N/Q95T/T199A/A517P/A642V,D12N/Q95T/A517P/L630M/V756C, D12N/R136Q,D12N/R136Q/R139K/L176T/A517P/G603Q/V756C,D12N/R136Q/R139K/L176T/L630M/V756C, D12N/R136Q/R139K/L176T/A642V,D12N/R136Q/R139K/T199A/R208K/A517P/L630M/A642V/V756C,D12N/R136Q/R139K/A517P, D12N/R136Q/R139K/A517P/L630M/A642V,D12N/R136Q/R139K/A517P/L630M/A642V/V756C, D12N/R136Q/R139K/A517P/V756C,D12N/R136Q/R139K/G603E/L630M, D12N/R136Q/R139K/G603Q/A642V/V756C,D12N/R136Q/R139K/L630M, D12N/R136Q/R139K/A642V/V756C,D12N/R136Q/L176T/G603Q/V756C,D12N/R136Q/T199A/R208K/A517P/G603E/A642V/V756C,D12N/R136Q/T199A/A517P/V756C, D12N/R136Q/R208K, D12N/R136Q/A517P/L630M,D12N/R136Q/G603E/A642V, D12N/R136Q/G603E/A642V/V756C,D12N/R136Q/G603Q/L630M/V756C, D12N/R136Q/G603Q/A642V/V756C,D12N/R136Q/G603Q/V756C, D12N/R139K/T199A/R208K/A642V,D12N/R139K/T199A/A517P/L630M/V756C, D12N/R139K/R208K/A517P,D12N/R139K/A517P/G603Q/A642V/V756C, D12N/R139K/G603E/A642V/V756C,D12N/R139K/G603Q/V756C, D12N/R139K/L630M/A642V,D12N/R139K/L630M/A642V/V756C, D12N/R139K/L630M/V756C,D12N/L176T/A517P/G603E/L630M, D12N/L176T/A517P/L630M/A642V/V756C,D12N/L176T/A517P/V756C, D12N/T199A/R208K/A517P/A642V,D12N/T199A/L630M/A642V/V756C, D12N/T199A/A642V/V756C,D12N/R208K/A517P/G603E/H623N/L630M/A642V, D12N/R208K/L630M/V756C,D12N/A517P/G603E/V756C, D12N/A517P/G603Q/L630M/V756C,D12N/A517P/L630M/A642V/V756C, D12N/G603E/A642V/V756C,D12N/G603Q/L630M/V756C, D12N/G603Q/A642V/V756C, D12N/G603Q/V756C,D12N/L630M/A642V/V756C, D12S/E45A/Q95T/R136Q/V756C,D12S/E45A/R136Q/R139K/R208K/G603E/L630M/V756C,D12S/E45A/R136Q/R139K/A517P/V756C,D12S/E45A/R136Q/L176T/A517P/G603E/L630M/A642V,D12S/E45A/R136Q/A517P/G603Q/A642V/V756C/A789V,D12S/E45A/R139K/L176T/R208K/A517P/G603E/L630M,D12S/E45A/R139K/A517P/V756C, D12S/E45A/R139K/V756C,D12S/E45A/L176T/G603Q/L630M/A642V/V756C,D12S/E45A/R208K/A517P/G603E/A642V/V756C,D12S/Q95T/R139K/A517P/L630M/V756C, D12S/Q95T/L630M/V756C,D12S/R136Q/R139K/L176T/A517P/G603Q/L630M, D12S/R136Q/R139K/L176T/V756C,D12S/R136Q/R139K/T199A/R208K/A517P/G603E/V756C,D12S/R136Q/R139K/R208K/A517P,D12S/R136Q/R139K/A517P/G603E/L630M/A642V/V756C,D12S/R136Q/R139K/A517P/G603E/L630M/V756C,D12S/R136Q/R139K/A517P/G603E/V756C,D12S/R136Q/R139K/A517P/G603Q/L630M/A642V/V756C,D12S/R136Q/R139K/A517P/G603Q/V756C, D12S/R136Q/R139K/A517P/L630M,D12S/R136Q/R139K/A517P/L630M/A642V, D12S/R136Q/R139K/G603Q/L630M/A642V,D12S/R136Q/R139K/G603Q/L630M/V756C, D12S/R136Q/R139K/L630M/A642V/V756C,D12S/R136Q/R139K/L630M/V756C,D12S/R136Q/L176T/R208K/A517P/G603E/L630M/V756C,D12S/R136Q/L176T/A517P/A642V, D12S/R136Q/T199A/R208K/L630M/A642V,D12S/R136Q/R208K/G603Q/A642V, D12S/R136Q/A517P/G603Q/L630M/A642V,D12S/R136Q/A517P/G603Q/L630M/V756C, D12S/R136Q/A517P/L630M/A642V,D12S/R136Q/A517P/A642V, D12S/R136Q/A517P/A642V/V756C,D12S/R136Q/A517P/V756C, D12S/R136Q/G603E/V756C,D12S/R136Q/G603Q/L630M/A642V/V756C, D12S/R136Q/L630M,D12S/R136Q/L630M/V756C, D12S/R139K/L176T/A517P/G603Q/L630M/V756C,D12S/R139K/L176T/L630M/V756C, D12S/R139K/R208K/A642V,D12S/R139K/A517P/L630M/A642V/V756C, D12S/R139K/A517P/A642V,D12S/R139K/G603Q/V756C, D12S/L176T/G603E/V756C,D12S/L176T/G603Q/L630M/V756C, D12S/T199A/V756C,D12S/R208K/G603Q/L630M/V756C, D12S/A517P/G603Q/L630M/A642V/V756C,D12S/A517P/L630M/A642V/V756C, D12S/A517P/A642V/V756C,D12S/G603Q/L630M/V756C, D12S/L630M/V756C, A25E/L176M/Q198R/S532Y/S539R,L116F/S142N/Q198R/Y434H/P440R,R136Q/R139K/L176T/T199A/R208K/A517P/L630M/A642V,R136Q/R139K/L176T/A517P/L630M/A642V/V756C,R136Q/R139K/T199A/A517P/G603E/V756C,R136Q/R139K/R208K/A517P/L630M/V756C,R136Q/R139K/R208K/G603Q/L630M/V756C,R136Q/R139K/A517P/G603E/A642V/V756C,R136Q/R139K/A517P/G603Q/L630M/A642V/V756C,R136Q/R139K/A517P/G603Q/V756C, R136Q/R139K/A517P/V756C,R136Q/R139K/G603Q, R136Q/R139K/L630M/A642V/V756C, R136Q/A517P/V756C,R136Q/G603Q/V756C, R136Q/L630M/A642V, R136Q/L630M/V756C, R136Q/A642V,R136Q/A642V/V756C, R136Q/V756C, R139K/T199A/R208K/A517P/L630M/V756C,R139K/T199A/A517P/A642V, R139K/R208K/A517P/L630M/A642V/V756C,R139K/A517P/G603Q/V756C, R139K/A517P/L630M/V756C, R139K/A642V/V756C,H154A/S532Y/G652R/H788K, T199A/A517P/G603Q/L630M/V756C,R208K/A517P/L630M/A642V/V756C, A517P/G603E/L630M/A642V/V756C,A517P/L630M/V756C, G603E/L630M/V756C, and G603Q/V756C, wherein thepositions are numbered with reference to SEQ ID NO: 8420. In someembodiments, the polypeptide sequence of the engineered sucrose synthasecomprises a sequence that is at least 90% identical to any of SEQ IDNOS: 8798, 8800, 8802, 8804, 8806, 8808, 8810, 8812, 8814, 8816, 8818,8820, 8822, 8824, 8826, 8828, 8830, 8832, 8834, 8836, 8838, 8840, 8842,8844, 8846, 8848, 8850, 8852, 8854, 8856, 8858, 8860, 8862, 8864, 8866,8868, 8870, 8872, 8874, 8876, 8878, 8880, 8882, 8884, 8886, 8888, 8890,8892, 8894, 8896, 8898, 8900, 8902, 8904, 8906, 8908, 8910, 8912, 8914,8916, 8918, 8920, 8922, 8924, 8926, 8928, 8930, 8932, 8934, 8936, 8938,8940, 8942, 8944, 8946, 8948, 8950, 8952, 8954, 8956, 8958, 8960, 8962,8964, 8966, 8968, 8970, 8972, 8974, 8976, 8978, 8980, 8982, 8984, 8986,8988, 8990, 8992, 8994, 8996, 8998, 9000, 9002, 9004, 9006, 9008, 9010,9012, 9014, 9016, 9018, 9020, 9022, 9024, 9026, 9028, 9030, 9032, 9034,9036, 9038, 9040, 9042, 9044, 9046, 9048, 9050, 9052, 9054, 9056, 9058,9060, 9062, 9064, 9066, 9068, 9070, 9072, 9074, 9076, 9078, 9080, 9082,9084, 9086, 9088, 9090, 9092, 9094, 9096, 9098, 9100, 9102, 9104, and9106. In some embodiments, the polypeptide sequence of the engineeredsucrose synthase comprises a sequence at least 95% identical to any ofSEQ ID NOS: 8798, 8800, 8802, 8804, 8806, 8808, 8810, 8812, 8814, 8816,8818, 8820, 8822, 8824, 8826, 8828, 8830, 8832, 8834, 8836, 8838, 8840,8842, 8844, 8846, 8848, 8850, 8852, 8854, 8856, 8858, 8860, 8862, 8864,8866, 8868, 8870, 8872, 8874, 8876, 8878, 8880, 8882, 8884, 8886, 8888,8890, 8892, 8894, 8896, 8898, 8900, 8902, 8904, 8906, 8908, 8910, 8912,8914, 8916, 8918, 8920, 8922, 8924, 8926, 8928, 8930, 8932, 8934, 8936,8938, 8940, 8942, 8944, 8946, 8948, 8950, 8952, 8954, 8956, 8958, 8960,8962, 8964, 8966, 8968, 8970, 8972, 8974, 8976, 8978, 8980, 8982, 8984,8986, 8988, 8990, 8992, 8994, 8996, 8998, 9000, 9002, 9004, 9006, 9008,9010, 9012, 9014, 9016, 9018, 9020, 9022, 9024, 9026, 9028, 9030, 9032,9034, 9036, 9038, 9040, 9042, 9044, 9046, 9048, 9050, 9052, 9054, 9056,9058, 9060, 9062, 9064, 9066, 9068, 9070, 9072, 9074, 9076, 9078, 9080,9082, 9084, 9086, 9088, 9090, 9092, 9094, 9096, 9098, 9100, 9102, 9104,and 9106. In some embodiments, the polypeptide sequence of theengineered sucrose synthase comprises any of SEQ ID NOS: 8798, 8800,8802, 8804, 8806, 8808, 8810, 8812, 8814, 8816, 8818, 8820, 8822, 8824,8826, 8828, 8830, 8832, 8834, 8836, 8838, 8840, 8842, 8844, 8846, 8848,8850, 8852, 8854, 8856, 8858, 8860, 8862, 8864, 8866, 8868, 8870, 8872,8874, 8876, 8878, 8880, 8882, 8884, 8886, 8888, 8890, 8892, 8894, 8896,8898, 8900, 8902, 8904, 8906, 8908, 8910, 8912, 8914, 8916, 8918, 8920,8922, 8924, 8926, 8928, 8930, 8932, 8934, 8936, 8938, 8940, 8942, 8944,8946, 8948, 8950, 8952, 8954, 8956, 8958, 8960, 8962, 8964, 8966, 8968,8970, 8972, 8974, 8976, 8978, 8980, 8982, 8984, 8986, 8988, 8990, 8992,8994, 8996, 8998, 9000, 9002, 9004, 9006, 9008, 9010, 9012, 9014, 9016,9018, 9020, 9022, 9024, 9026, 9028, 9030, 9032, 9034, 9036, 9038, 9040,9042, 9044, 9046, 9048, 9050, 9052, 9054, 9056, 9058, 9060, 9062, 9064,9066, 9068, 9070, 9072, 9074, 9076, 9078, 9080, 9082, 9084, 9086, 9088,9090, 9092, 9094, 9096, 9098, 9100, 9102, 9104, and 9106.

The present invention also provides engineered glycosyltransferases thatare NDP-glycosyltransferases, wherein the engineeredNDP-glycosyltransferase are NDP-glycosyltransferases selected fromADP-glucose-dependent glycosyltransferases (AGTs), CDP-glucose-dependentglycosyltransferases (CGTs), GDP-glucose-dependent glycosyltransferase(GGTs), TDP-glucose-dependent glycosyltransferases (TGTs), andIDP-glucose-dependent glycosyltransferase (IGTs). In some embodiments,the engineered NDP-glycosyltransferase is an ADP-glucose-dependentglycosyltransferase. In some embodiments, the engineeredNDP-glycotransferase is not an UDP-glucose-dependentglycosyltransferase.

The present invention also provides engineered polynucleotides encodingat least one engineered glycosyltransferase polypeptide provided herein.In some embodiments, the engineered polynucleotide encoding at least oneengineered glycosyltransferase provided herein comprises at least 60%,65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99%, or more sequence identity to SEQ ID NO: 5, 7,9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43,45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 75, 77, 79, 81, 83,85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115,117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143,145, 147, 149, 151, 153, 155, 157, 159, 161, 163, 165, 167, 169, 171,173, 175, 177, 179, 181, 183, 185, 187, 189, 191, 193, 195, 197, 199,201, 203, 205, 207, 209, 211, 213, 215, 217, 219, 221, 223, 225, 227,229, 231, 233, 235, 237, 239, 241, 243, 245, 247, 249, 251, 253, 255,257, 259, 261, 263, 265, 267, 269, 271, 273, 275, 277, 279, 281, 283,285, 287, 289, 291, 293, 295, 297, 299, 301, 303, 305, 307, 309, 311,313, 315, 317, 319, 321, 323, 325, 327, 329, 331, 333, 335, 337, 339,341, 343, 345, 347, 349, 351, 353, 355, 357, 359, 361, 363, 365, 367,369, 371, 373, 375, 377, 379, 381, 383, 385, 387, 389, 391, 393, 395,397, 399, 401, 403, 405, 407, 409, 411, 413, 415, 417, 419, 421, 423,425, 427, 429, 431, 433, 435, 437, 439, 441, 443, 445, 447, 449, 451,453, 455, 457, 459, 461, 463, 465, 467, 469, 471, 473, 475, 477, 479,481, 483, 485, 487, 489, 491, 493, 495, 497, 499, 501, 503, 505, 507,509, 511, 513, 515, 517, 519, 521, 523, 525, 527, 529, 531, 533, 535,537, 539, 541, 543, 545, 547, 549, 551, 553, 555, 557, 559, 561, 563,565, 567, 569, 571, 573, 575, 577, 579, 581, 583, 585, 587, 589, 591,593, 595, 597, 599, 601, 603, 605, 607, 609, 611, 613, 615, 617, 619,621, 623, 625, 627, 629, 631, 633, 635, 637, 639, 641, 643, 645, 647,649, 651, 653, 655, 657, 659, 661, 663, 665, 667, 669, 671, 673, 675,677, 679, 681, 683, 685, 687, 689, 691, 693, 695, 697, 699, 701, 703,705, 707, 709, 711, 713, 715, 717, 719, 721, 723, 725, 727, 729, 731,733, 735, 737, 739, 741, 743, 745, 747, 749, 751, 753, 769, 771, 773,775, 777, 779, 781, 783, 785, 787, 789, 791, 793, 795, 797, 799, 801,803, 805, 807, 809, 811, 813, 815, 817, 819, 821, 823, 825, 827, 829,831, 833, 835, 837, 839, 841, 843, 845, 847, 849, 851, 853, 855, 857,859, 861, 863, 865, 867, 869, 871, 873, 875, 877, 879, 881, 883, 885,887, 889, 891, 893, 895, 897, 899, 901, 903, 905, 907, 909, 911, 913,915, 917, 919, 921, 923, 925, 927, 929, 931, 933, 935, 937, 939, 941,943, 945, 947, 949, 951, 953, 955, 957, 959, 961, 963, 965, 967, 969,971, 973, 975, 977, 979, 981, 983, 985, 987, 989, 991, 993, 995, 997,999, 1001, 1003, 1005, 1007, 1009, 1011, 1013, 1015, 1017, 1019, 1021,1023, 1025, 1027, 1029, 1031, 1033, 1035, 1037, 1039, 1041, 1043, 1045,1047, 1049, 1051, 1053, 1055, 1057, 1059, 1061, 1063, 1065, 1067, 1069,1071, 1073, 1075, 1077, 1289, 1291, 1293, 2595, 2597, 2599, 2601, 2603,2605, 2607, 2609, 2611, 2613, 2615, 2617, 2619, 2621, 2623, 2625, 2627,2629, 2631, 2633, 2635, 2637, 2639, 2641, 2643, 2645, 2647, 2649, 2651,2653, 2655, 2657, 2659, 2661, 2663, 2665, 2667, 2669, 2671, 2673, 2675,2677, 2679, 2681, 2683, 2685, 2687, 2689, 2691, 2693, 2695, 2697, 2699,2701, 2703, 2705, 2707, 2709, 2711, 2713, 2715, 2717, 2719, 2721, 2723,2725, 2727, 2729, 2731, 2733, 2735, 2737, 2739, 2741, 2743, 2745, 2747,2749, 2751, 2753, 2755, 2757, 2759, 2761, 2763, 2765, 2767, 2769, 2771,2773, 2775, 2777, 2779, 2781, 2783, 2785, 2787, 2789, 2791, 2793, 2795,2797, 2799, 2801, 2803, 2805, 2807, 2809, 2811, 2813, 2815, 2817, 2819,2821, 2823, 2825, 2827, 2829, 2831, 2833, 2835, 2837, 2839, 2841, 2843,2845, 2847, 2849, 2851, 2823, 2855, 2857, 2859, 2861, 2863, 2865, 2867,2869, 2871, 2873, 2875, 2877, 2879, 2881, 2883, 2885, 2887, 2889, 2891,2893, 2895, 2897, 2899, 2901, 2903, 2905, 2907, 2909, 2911, 2913, 2915,2917, 2919, 2921, 2923, 2925, 2927, 2829, 2931, 2933, 2935, 2937, 2939,2941, 2943, 2945, 2947, 2949, 2951, 2953, 2955, 2957, 2959, 2961, 2963,2965, 2967, 2969, 2971, 2973, 2975, 2977, 2979, 2981, 2983, 2985, 2987,2989, 2991, 2993, 2995, 2997, 2999, 3001, 3003, 3005, 3007, 3009, 3011,3013, 3015, 3017, 3019, 3021, 3023, 3025, 3027, 3029, 3031, 3033, 3035,3037, 3039, 3041, 3043, 3045, 3047, 3049, 3051, 3053, 3055, 3057, 3059,3061, 3063, 3065, 3067, 3069, 3071, 3073, 3075, 3077, 3079, 3081, 3083,3085, 3087, 3089, 3091, 3093, 3095, 3097, 3099, 3101, 3103, 3105, 3107,3109, 3111, 3113, 3115, 3117, 3119, 3121, 3123, 3125, 3127, 3129, 3131,3133, 3135, 3137, 3139, 3141, 3143, 3145, 3147, 3149, 3151, 3153, 3155,3157, 3159, 3161, 3163, 3165, 3167, 3169, 3171, 3173, 3175, 3177, 3179,3181, 3183, 3185, 3187, 3189, 3191, 3193, 3195, 3197, 3199, 3201, 3203,3205, 3207, 3209, 3211, 3213, 3215, 3217, 3219, 3221, 3223, 3225, 3227,3229, 3231, 3233, 3235, 3237, 3239, 3241, 3243, 3245, 3247, 3249, 3251,3253, 3255, 3257, 3259, 3261, 3263, 3265, 3267, 3269, 3271, 3273, 3275,3277, 3279, 3281, 3283, 3285, 3287, 3289, 3291, 3293, 3295, 3297, 3299,3301, 3303, 3305, 3307, 3309, 3311, 3313, 3315, 3317, 3319, 3321, 3323,3325, 3327, 3329, 3331, 3333, 3335, 3337, 3339, 3341, 3343, 3345, 3347,3349, 3351, 3353, 3355, 3357, 3359, 3361, 3363, 3365, 3367, 3369, 3371,3373, 3375, 3377, 3379, 3381, 3383, 3385, 3387, 3389, 3391, 3393, 3395,3397, 3399, 3401, 3403, 3405, 3407, 3409, 3411, 3413, 3415, 3417, 3419,3421, 3423, 3425, 3427, 3429, 3431, 3433, 3435, 3437, 3439, 3441, 3443,3445, 3447, 3449, 3451, 3453, 3455, 3457, 3459, 3461, 3463, 3465, 3467,3469, 3471, 3473, 3475, 3477, 3479, 3481, 3483, 3485, 3487, 3489, 3491,3493, 3495, 3497, 3499, 3501, 3503, 3505, 3507, 3509, 3511, 3513, 3515,3517, 3519, 3521, 3523, 3525, 3527, 3529, 3531, 3533, 3535, 3537, 3539,3541, 3543, 3545, 3547, 3549, 3551, 3553, 3555, 3557, 3559, 3561, 3563,3565, 3567, 3569, 3571, 3573, 3575, 3577, 3579, 3581, 3583, 3585, 3587,3589, 3591, 3593, 3595, 3597, 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7981,7983, 7985, 7987, 7989, 7991, 7993, 7995, 7997, 7999, 8001, 8003, 8005,8007, 8009, 8011, 8013, 8015, 8017, 8019, 8021, 8023, 8025, 8027, 8029,8031, 8033, 8035, 8037, 8039, 8041, 8043, 8045, 8047, 8049, 8051, 8053,8055, 8057, 8059, 8061, 8063, 8065, 8067, 8069, 8071, 8073, 8075, 8077,8079, 8081, 8083, 8085, 8087, 8089, 8091, 8093, 8095, 8097, 8099, 8101,8103, 8105, 8107, 8109, 8111, 8113, 8115, 8117, 8119, 8121, 8123, 8125,8127, 8129, 8131, 8133, 8135, 8137, 8139, 8141, 8143, 8145, 8147, 8149,8151, 8153, 8155, 8157, 8159, 8161, 8163, 8165, 8167, 8169, 8171, 8173,8175, 8177, 8179, 8181, 8183, 8185, 8187, 8189, 8191, 8193, 8195, 8197,8199, 8201, 8203, 8205, 8207, 8209, 8211, 8213, 8215, 8217, 8219, 8221,8223, 8225, 8227, 8229, 8231, 8233, 8235, 8237, 8239, 8241, 8243, 8245,8247, 8249, 8251, 8253, 8255, 8257, 8259, 8261, 8263, 8265, 8267, 8269,8271, 8273, 8275, 8277, 8279, 8281, 8283, 8285, 8287, 8289, 8291, 8293,8295, 8297, 8299, 8301, 8303, 8305, 8307, 8309, 8311, 8313, 8315, 8317,8319, 8321, 8323, 8325, 8327, 8329, 8331, 8333, 8335, 8337, 8339, 8341,8343, 8345, 8347, 8349, 8351, 8353, 8355, 8357, 8359, 8361, 8363, 8365,8367, 8481, 8483, 8485, 8487, 8489, 8491, 8493, 8495, 8497, 8489, 8501,8503, 8505, 8507, 8509, 8511, 8513, 8515, 8517, 8519, 8521, 8523, 8525,8527, 8529, 8531, 8533, 8535, 8537, 8539, 8541, 8543, 8545, 8547, 8549,8551, 8553, 8555, 8557, 8559, 8561, 8563, 8565, 8567, 8569, 8571, 8573,8575, 8577, 8579, 8581, 8583, 8585, 8587, 8589, 8591, 8593, 8595, 8597,8599, 8601, 8603, 8605, 8607, 8609, 8611, 8613, 8615, 8617, 8619, 8621,8623, 8625, 8627, 8629, 8631, 8633, 8635, 8637, 8639, 8641, 8643, 8645,8647, 8649, 8651, 8653, 8655, 8657, 8659, 8661, 8663, 8665, 8667, 8669,8671, 8673, 8675, 8677, 8679, 8681, 8683, 8685, 8687, 8689, 8691, 8693,8695, 8697, 8699, 8701, 8703, 8705, 8707, 8709, 8711, 8713, 8715, 8717,8719, 8721, 8723, 8725, 8727, 8729, 8731, 8733, 8735, 8737, 8739, 8741,8743, 8745, 8747, 8749, 8751, 8753, 8755, 8757, 8759, 8761, 8763, 8765,8767, 8769, 8771, 8773, 8775, 8777, 8779, 8781, 8783, 8785, 8787, 8789,8791, 8793, 8795, 9107, 9109, 9111, 9113, 9115, 9117, 9119, 9121, 9123,9125, 9127, 9129, 9131, 9133, 9135, 9137, 9139, 9141, 9143, 9145, 9147,9149, 9151, 9153, 9155, 9157, 9159, 9161, 9163, 9165, 9167, 9169, 9171,9173, 9175, 9177, 9179, 9181, 9183, 9185, 9187, 9189, 9191, 9193, 9195,9197, 9199, 9201, 9203, 9205, 9207, 9209, 9211, 9213, 9215, 9217, 9219,9221, 9223, 9225, 9227, 9229, 9231, 9233, 9235, 9237, and/or 9239.

The present invention also provides engineered polynucleotides encodingat least one engineered sucrose synthase polypeptide provided herein. Insome embodiments, the engineered polynucleotide encoding at least oneengineered sucrose synthase provided herein comprises at least 60%, 65%,70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99%, or more sequence identity to SEQ ID NO: 1079, 1081,1083, 1085, 1087, 1089, 1091, 1093, 1095, 1097, 1099, 1101, 1103, 1105,1107, 1109, 1111, 1113, 1115, 1117, 1119, 1121, 1123, 1125, 1127, 1129,1131, 1133, 1135, 1137, 1139, 1141, 1143, 1145, 1147, 1149, 1151, 1153,1155, 1157, 1159, 1161, 1163, 1165, 1167, 1169, 1171, 1173, 1175, 1177,1179, 1181, 1183, 1185, 1187, 1189, 1191, 1193, 1195, 1197, 1199, 1201,1203, 1205, 1207, 1209, 1211, 1213, 1215, 1217, 1219, 1221, 1223, 1225,1227, 1229, 1231, 1233, 1235, 1237, 1239, 1241, 1243, 1245, 1247, 1249,1251, 1253, 1255, 1257, 1259, 1261, 1263, 1265, 1267, 1269, 1271, 1273,1275, 1277, 1279, 1281, 1283, 1285, 1287, 1295, 1297, 1299, 1301, 1303,1305, 1307, 1309, 1311, 1313, 1315, 1317, 1319, 1321, 1323, 1325, 1327,1329, 1331, 1333, 1335, 1337, 1339, 1341, 1343, 1345, 1347, 1349, 1351,1353, 1355, 1357, 1359, 1361, 1363, 1365, 1367, 1369, 1371, 1373, 1375,1377, 1379, 1381, 1383, 1385, 1387, 1389, 1391, 1393, 1395, 1397, 1399,1401, 1403, 1405, 1407, 1409, 1411, 1413, 1415, 1417, 1419, 1421, 1423,1425, 1427, 1429, 1431, 1433, 1435, 1437, 1439, 1441, 1443, 1445, 1447,1449, 1451, 1453, 1455, 1457, 1459, 1461, 1463, 1465, 1467, 1469, 1471,1473, 1475, 1477, 1479, 1481, 1483, 1485, 1487, 1489, 1491, 1493, 1495,1497, 1499, 1501, 1503, 1505, 1507, 1509, 1511, 1513, 1515, 1517, 1519,1521, 1523, 1525, 1527, 1529, 1531, 1533, 1535, 1537, 1539, 1541, 1543,1545, 1547, 1549, 1551, 1553, 1555, 1557, 1559, 1561, 1563, 1565, 1567,1569, 1571, 1573, 1575, 1577, 1579, 1581, 1583, 1585, 1587, 1589, 1591,1593, 1595, 1597, 1599, 1601, 1603, 1605, 1607, 1609, 1611, 1613, 1615,1617, 1619, 1621, 1623, 1625, 1627, 1629, 1631, 1633, 1635, 1637, 1639,1641, 1643, 1645, 1647, 1649, 1651, 1653, 1655, 1657, 1659, 1661, 1663,1665, 1667, 1669, 1671, 1673, 1675, 1677, 1679, 1681, 1683, 1685, 1687,1689, 1691, 1693, 1695, 1697, 1699, 1701, 1703, 1705, 1707, 1709, 1711,1713, 1715, 1717, 1719, 1721, 1723, 1725, 1727, 1729, 1731, 1733, 1735,1737, 1739, 1741, 1743, 1745, 1747, 1749, 1751, 1753, 1755, 1757, 1759,1761, 1763, 1765, 1767, 1769, 1771, 1773, 1775, 1777, 1779, 1781, 1783,1785, 1787, 1789, 1791, 1793, 1795, 1797, 1799, 1801, 1803, 1805, 1807,1809, 1811, 1813, 1815, 1817, 1819, 1821, 1823, 1825, 1827, 1829, 1831,1833, 1835, 1837, 1839, 1841, 1843, 1845, 1847, 1849, 1851, 1853, 1855,1857, 1859, 1861, 1863, 1865, 1867, 1869, 1871, 1873, 1875, 1877, 1879,1881, 1883, 1885, 1887, 1889, 1891, 1893, 1895, 1897, 1899, 1901, 1903,1905, 1907, 1909, 1911, 1913, 1915, 1917, 1919, 1921, 1923, 1925, 1927,1929, 1931, 1933, 1935, 1937, 1939, 1941, 1943, 1945, 1947, 1949, 1951,1953, 1955, 1957, 1959, 1961, 1963, 1965, 1967, 1969, 1971, 1973, 1975,1977, 1979, 1981, 1983, 1985, 1987, 1989, 1991, 1993, 1995, 1997, 1999,2001, 2003, 2005, 2007, 2009, 2011, 2013, 2015, 2017, 2019, 2021, 2023,2025, 2027, 2029, 2031, 2033, 2035, 2037, 2039, 2041, 2043, 2045, 2047,2049, 2051, 2053, 2055, 2057, 2059, 2061, 2063, 2065, 2067, 2069, 2071,2073, 2075, 2077, 2079, 2081, 2083, 2085, 2087, 2089, 2091, 2093, 2095,2097, 2099, 2101, 2103, 2105, 2107, 2109, 2111, 2113, 2115, 2117, 2119,2121, 2123, 2125, 2127, 2129, 2131, 2133, 2135, 2137, 2139, 2141, 2143,2145, 2147, 2149, 2151, 2153, 2155, 2157, 2159, 2161, 2163, 2165, 2167,2169, 2171, 2173, 2175, 2177, 2179, 2181, 2183, 2185, 2187, 2189, 2191,2193, 2195, 2197, 2199, 2201, 2203, 2205, 2207, 2209, 2211, 2213, 2215,2217, 2219, 2221, 2223, 2225, 2227, 2229, 2231, 2233, 2235, 2237, 2239,2241, 2243, 2245, 2247, 2249, 2251, 2253, 2255, 2257, 2259, 2261, 2263,2265, 2267, 2269, 2271, 2273, 2275, 2277, 2279, 2281, 2283, 2285, 2287,2289, 2291, 2293, 2295, 2297, 2299, 2301, 2303, 2305, 2307, 2309, 2311,2313, 2315, 2317, 2319, 2321, 2323, 2325, 2327, 2329, 2331, 2333, 2335,2337, 2339, 2341, 2343, 2345, 2347, 2349, 2351, 2353, 2355, 2357, 2359,2361, 2363, 2365, 2367, 2369, 2371, 2373, 2375, 2377, 2379, 2381, 2383,2385, 2387, 2389, 2391, 2393, 2395, 2397, 2399, 2401, 2403, 2405, 2407,2409, 2411, 2413, 2415, 2417, 2419, 2421, 2423, 2425, 2427, 2429, 2431,2433, 2435, 2437, 2439, 2441, 2443, 2445, 2447, 2449, 2451, 2453, 2455,2457, 2459, 2461, 2463, 2465, 2467, 2469, 2471, 2473, 2475, 2477, 2479,2481, 2483, 2485, 2487, 2489, 2491, 2493, 2495, 2497, 2499, 2501, 2503,2505, 2507, 2509, 2511, 2513, 2515, 2517, 2519, 2521, 2523, 2525, 2527,2529, 2531, 2533, 2535, 2537, 2539, 2541, 2543, 2545, 2547, 2549, 2551,2553, 2555, 2557, 2559, 2561, 2563, 2565, 2567, 2569, 2571, 2573, 2575,2577, 2579, 2581, 2583, 2585, 2587, 2589, 2591, 2593, 7437, 7439, 7441,7443, 7445, 7447, 7449, 7451, 7453, 7455, 7457, 7459, 7461, 7463, 7465,7467, 7469, 7471, 7473, 7475, 7477, 7479, 7481, 7483, 7485, 7487, 7489,7491, 7493, 7495, 7497, 7499, 7501, 7503, 7505, 7507, 7509, 7511, 7513,7515, 7517, 7519, 7521, 7523, 7525, 7527, 7529, 7531, 7533, 7535, 7537,7539, 7541, 7543, 7545, 7547, 7549, 7551, 7553, 7555, 7557, 7559, 7561,7563, 7565, 7567, 7569, 7571, 7573, 7575, 7577, 7579, 7581, 7583, 7585,7587, 7589, 7591, 7593, 7595, 7597, 7599, 7601, 7603, 7605, 7607, 7609,7611, 7613, 7615, 7617, 7619, 7621, 7623, 7625, 7627, 7629, 7631, 7633,7635, 7637, 7639, 7641, 7643, 7645, 7647, 7649, 7651, 7653, 7655, 7657,7659, 7661, 7663, 7665, 7667, 7669, 7671, 7673, 7675, 7677, 7679, 7681,7683, 7685, 7687, 7689, 7691, 7693, 7695, 7697, 7699, 7701, 7703, 7705,7707, 7709, 7711, 7713, 7715, 7717, 7719, 7721, 7723, 7725, 7727, 7729,7731, 7733, 7735, 7737, 7739, 7741, 7743, 7745, 7747, 7749, 7751, 7753,7755, 7757, 7759, 7761, 7763, 8369, 8371, 8373, 8375, 8377, 8379, 8381,8383, 8385, 8387, 8389, 8391, 8393, 8395, 8397, 8399, 8401, 8403, 8405,8407, 8409, 8411, 8413, 8415, 8417, 8419, 8421, 8423, 8425, 8427, 8429,8431, 8433, 8435, 8437, 8439, 8441, 8443, 8445, 8447, 8449, 8451, 8453,8455, 8457, 8459, 8461, 8463, 8465, 8467, 8469, 8471, 8473, 8475, 8477,8479, 8797, 8799, 8801, 8803, 8805, 8807, 8809, 8811, 8813, 8815, 8817,8819, 8821, 8823, 8825, 8827, 8829, 8831, 8833, 8835, 8837, 8839, 8841,8843, 8845, 8847, 8849, 8851, 8853, 8855, 8857, 8859, 8861, 8863, 8865,8867, 8869, 8871, 8873, 8875, 8877, 8879, 8881, 8883, 8885, 8887, 8889,8891, 8893, 8895, 8897, 8899, 8901, 8903, 8905, 8907, 8909, 8911, 8913,8915, 8917, 8919, 8921, 8923, 8925, 8927, 8929, 8931, 8933, 8935, 8937,8939, 8941, 8943, 8945, 8947, 8949, 8951, 8953, 8955, 8957, 8959, 8961,8963, 8965, 8967, 8969, 8971, 8973, 8975, 8977, 8979, 8981, 8983, 8985,8987, 8989, 8991, 8993, 8995, 8997, 8999, 9001, 9003, 9005, 9007, 9009,9011, 9013, 9015, 9017, 9019, 9021, 9023, 9025, 9027, 9029, 9031, 9033,9035, 9037, 9039, 9041, 9043, 9045, 9047, 9049, 9051, 9053, 9055, 9057,9059, 9061, 9063, 9065, 9067, 9069, 9071, 9073, 9075, 9077, 9079, 9081,9083, 9085, 9087, 9089, 9091, 9093, 9095, 9097, 9099, 9101, 9103, and/or9105.

The present invention also provides vectors comprising at least oneengineered polynucleotide provided herein. In some embodiments, thevectors further comprise at least one control sequence.

The present invention also provides host cells comprising at least oneengineered polypeptide provided herein. In some embodiments, the hostcells comprise at least one vector provided herein.

In some embodiments, the host cell is selected from eukaryotic andprokaryotic organisms. In some additional embodiments, the host cell isE. coli.

The present invention also provides methods for producing at least oneengineered glycosyltransferase variant provided herein, comprisingculturing the host cell provided herein, under conditions such that theengineered glycosyltransferase variant is produced by the host cell. Insome embodiments, the methods further comprise the step of recoveringthe engineered glycosyltransferase variant.

The present invention also provides compositions comprising at least oneengineered glycosyltransferase variant and/or sucrose synthase variantprovided herein. In some embodiments, the compositions comprise at leastone engineered glycosyltransferase variant provided herein.

The present invention also provides methods for producing at least oneengineered sucrose synthase variant provided herein, comprisingculturing the host cell provided herein, under conditions such that theengineered sucrose synthase variant is produced by the host cell. Insome embodiments, the methods further comprise the step of recoveringthe engineered sucrose synthase variant. In some embodiments, thecompositions comprise at least one engineered sucrose synthase variantprovided herein.

The present invention also provides methods for glycosylation of asubstrate comprising: providing at least one substrate, at least oneengineered glycosyl transferase selected from the even numberedsequences provided herein; contacting the substrate with theglycosyltransferase under conditions such that the substrate isglycosylated to produce at least one glycosylated product. In someembodiments, the substrate comprises at least one steviol glycoside. Insome embodiments, the glycosylated product comprises at least onemono-glycosylated and/or polyglycosylated product. It is not intendedthat the present invention be limited to any limitations regarding theextent of glycosylation of the product (e.g., diglycosylated,triglycosylated, and products with higher glycosylation levels find usein the present invention).

The present invention provides methods for producing rebaudioside M,comprising providing a rebaudioside D and/or rebaudioside I substrate,NDP-glucose, and a least one engineered glycosyltransferase providedherein, combining the rebaudioside D and rebaudioside I substrate,NDP-glucose, and the glycosyltransferase under conditions such thatrebaudioside M is produced. In some additional embodiments, the presentinvention provides methods for producing rebaudioside M, comprisingproviding a rebaudioside D substrate, NDP-glucose, and a least oneengineered glycosyltransferase provided herein, combining therebaudioside D substrate, NDP-glucose, and glycosyltransferase underconditions such that rebaudioside M is produced. In some furtherembodiments, the present invention provides methods for producingrebaudioside M, comprising providing a rebaudioside I substrate,NDP-glucose, and a least one engineered glycosyltransferase providedherein, combining the rebaudioside I substrate, NDP-glucose, andglycosyltransferase under conditions such that rebaudioside M isproduced. In some of the present embodiments, the NDP-glucose isselected from ADP-glucose, CDP-glucose, TDP-glucose, GDP-glucose, and/orIDT-glucose. In some additional embodiments, the NDP-glucose is notUDP-glucose.

The present invention provides methods for producing rebaudioside Aand/or rebaudioside I, comprising providing a stevioside substrate,NDP-glucose, and at least one engineered glycosyltransferase providedherein, combining the stevioside substrate, NDP-glucose, andglycosyltransferase under conditions such that rebaudioside A and/orrebaudioside I is produced. In some embodiments, the present inventionprovides methods for producing rebaudioside A, comprising providing astevioside substrate, NDP-glucose, and at least one engineeredglycosyltransferase provided herein, combining the stevioside substrate,NDP-glucose, and glycosyltransferase under conditions such thatrebaudioside A is produced. In some additional embodiments, the presentinvention provides methods for producing rebaudioside I, comprisingproviding a stevioside substrate, NDP-glucose, and at least oneengineered glycosyltransferase provided herein, combining the steviosidesubstrate, NDP-glucose, and glycosyltransferase under conditions suchthat rebaudioside I is produced. In some of the present embodiments, theNDP-glucose is selected from ADP-glucose, CDP-glucose, TDP-glucose,GDP-glucose, and/or IDT-glucose. In some additional embodiments, theNDP-glucose is not UDP-glucose.

The present invention also provides methods for producing rebaudiosideD, comprising providing a stevioside substrate, NDP-glucose, and atleast one engineered glycosyltransferase provided herein, combining thestevioside substrate, NDP-glucose, and glycosyltransferase underconditions such that rebaudioside D is produced. In some of the presentembodiments, the NDP-glucose is selected from ADP-glucose, CDP-glucose,TDP-glucose, GDP-glucose, and/or IDT-glucose. In some additionalembodiments, the NDP-glucose is not UDP-glucose.

The present invention also provides methods for producing rebaudioside Mcomprising providing a rebaudioside D and/or rebaudioside I substrate,ADP-glucose, and at least one engineered glycosyltransferase providedherein, combining the rebaudioside D and/or rebaudioside I substrate,ADP-glucose, and glycosyltransferase under conditions such thatrebaudioside M is produced. In some embodiments, the present inventionprovides methods for producing rebaudioside M comprising providing arebaudioside D substrate, ADP-glucose, and at least one engineeredglycosyltransferase of provided herein, combining the rebaudioside Dsubstrate, ADP-glucose, and glycosyltransferase under conditions suchthat rebaudioside M is produced. In some additional embodiments, thepresent invention provides methods for producing rebaudioside Mcomprising providing a rebaudioside I substrate, ADP-glucose, and atleast one engineered glycosyltransferase provided herein, combining therebaudioside I substrate, ADP-glucose, and glycosyltransferase underconditions such that rebaudioside M is produced. In some of the presentembodiments, the engineered glycosyltransferase comprises anADP-glycosyltransferase.

The present invention also provides methods for producing rebaudioside Aand/or rebaudioside I, comprising providing a stevioside substrate,ADP-glucose, and at least one engineered glycosyltransferase providedherein, combining the stevioside substrate, ADP-glucose, andglycosyltransferase under conditions such that rebaudioside A and/orrebaudioside I is produced. In some embodiments, the present inventionprovides methods for producing rebaudioside A, comprising providing astevioside substrate, ADP-glucose, and at least one engineeredglycosyltransferase provided herein, combining the stevioside substrate,ADP-glucose, and glycosyltransferase under conditions such thatrebaudioside A is produced. In some additional embodiments, the presentinvention provides methods for producing rebaudioside I, comprisingproviding a stevioside substrate, ADP-glucose, and at least oneengineered glycosyltransferase provided herein, combining the steviosidesubstrate, ADP-glucose, and glycosyltransferase under conditions suchthat rebaudioside I is produced. In some of the present embodiments, theengineered glycosyltransferase comprises an ADP-glycosyltransferase.

The present invention also provides methods for producing rebaudiosideD, comprising providing a stevioside substrate, ADP-glucose, and atleast one engineered glycosyltransferase provided herein, combining thestevioside substrate, ADP-glucose, and glycosyltransferase underconditions such that rebaudioside D is produced. In some of the presentembodiments, the engineered glycosyltransferase comprises anADP-glycosyltransferase.

The present invention also provides methods for producing rebaudioside Mcomprising providing a rebaudioside D substrate, NDP, sucrose, a sucrosesynthase, and at least one engineered glycosyltransferase providedherein, combining the rebaudioside D substrate, NDP, sucrose, sucrosesynthase, and glycosyltransferase under conditions such thatrebaudioside M is produced. In some of the present embodiments, theengineered glycosyltransferase comprises an ADP-glycosyltransferase.

In some additional embodiments, the NDP is selected from ADP, CDP, TDP,GDP, and/or IDT. In some additional embodiments, the NDP is not UDP.

The present invention also provides methods for producing rebaudioside Aand/or rebaudioside I comprising providing a stevioside substrate, NDP,sucrose, a sucrose synthase, and at least one engineeredglycosyltransferase provided herein, combining the stevioside substrate,NDP, sucrose, sucrose synthase, and glycosyltransferase under conditionssuch that rebaudioside A and/or rebaudioside I is produced. In someembodiments, the present invention provides methods for producingrebaudioside A, comprising providing a stevioside substrate, NDP,sucrose, a sucrose synthase, and at least one engineeredglycosyltransferase provided herein, combining the stevioside substrate,NDP, sucrose, sucrose synthase, and glycosyltransferase under conditionssuch that rebaudioside A is produced. In some additional embodiments,the present invention provides methods for producing rebaudioside I,comprising providing a stevioside substrate, NDP, sucrose, a sucrosesynthase, and at least one engineered ADP-glycosyltransferase providedherein, combining the stevioside substrate, NDP, sucrose, sucrosesynthase and an ADP-glycosyltransferase under conditions such thatrebaudioside I is produced. In some of the present embodiments, theengineered glycosyltransferase comprises an ADP-glycosyltransferase. Insome additional embodiments, the NDP is selected from ADP, CDP, TDP,GDP, and/or IDT. In some additional embodiments, the NDP is not UDP.

The present invention also provides methods for producing rebaudiosideD, comprising providing a stevioside substrate, NDP, sucrose, a sucrosesynthase, and at least one engineered glycosyltransferase providedherein, combining the stevioside substrate, NDP, sucrose, sucrosesynthase, and glycosyltransferase under conditions such thatrebaudioside D is produced. In some of the present embodiments, theengineered glycosyltransferase comprises an ADP-glycosyltransferase.

In some additional embodiments, the NDP is selected from ADP, CDP, TDP,GDP, and/or IDT. In some additional embodiments, the NDP is not UDP.

The present invention also provides methods for producing rebaudiosideM, comprising providing a stevioside substrate comprising at least onestevioside and/or a mixture of steviosides and rebA, NDP, sucrose, asucrose synthase, and at least one engineered glycosyltransferaseprovided herein, combining the stevioside substrate, NDP, sucrose,sucrose synthase, and glycosyltransferase under conditions such thatrebaudioside M is produced. In some of the present embodiments, theengineered glycosyltransferase comprises an ADP-glycosyltransferase. Insome additional embodiments, the NDP is selected from ADP, CDP, TDP,GDP, and/or IDT. In some additional embodiments, the NDP is not UDP.

The present invention also provides methods for producing rebaudiosideM, comprising providing a stevioside substrate, NDP, sucrose, at leastone sucrose synthase, and at least one engineered glycosyltransferase oprovided herein, combining the stevioside substrate, NDP, andglycosyltransferase under conditions such that rebaudioside A is firstproduced, rebaudioside D and/or rebaudioside I is then produced, andrebaudioside M finally produced. In some of the present embodiments, theengineered glycosyltransferase comprises an ADP-glycosyltransferase. Insome additional embodiments, the NDP is selected from ADP, CDP, TDP,GDP, and/or IDT. In some additional embodiments, the NDP is not UDP.

In some embodiments of the methods of the present invention for theproduction of rebaudioside(s), the methods are conducted as one-potreactions. In some additional embodiments, the methods are conductedsequentially. In some additional embodiments, the methods furthercomprise repeating the steps of the methods. In some furtherembodiments, sucrose is recycled during repeated steps. In someadditional embodiments, at least one engineered glycosyltransferaseand/or other reaction components are recycled. In some additionalembodiments, the stevioside substrate is extracted from Steviarebaudiana, while in some alternative embodiments, the steviosidesubstrate is synthetically produced, and still some further embodiments,the stevioside substrate is a mixture of steviosides that are eithernaturally and/or synthetically produced. In some further embodiments ofthe methods, at least one engineered glycosyltransferase is immobilized.In some additional embodiments of the methods, sucrose synthase isimmobilized. In still some additional embodiments, at least oneglycosyltransferase and/or the sucrose synthase is immobilized. In somefurther embodiments of the methods, a reaction product that includesfructose is produced. In some embodiments, the fructose is removed fromthe reaction product. In yet some additional embodiments, the methodsfurther comprises a washing step. In some embodiments, the washing stepcomprises exposing the rebaudioside M, rebaudioside A, rebaudioside I,and/or rebaudioside D produced from the method to a solvent. In someembodiments, the solvent is water. In still further embodiments, themethods further comprise at least one column chromatography step. Insome embodiments, at least one column chromatography step is conductedon the rebaudioside M, rebaudioside A, rebaudioside I, and/orrebaudioside D produced from the method. In some additional embodimentsof the methods, the at least one engineered glycosyltransferase is abeta-1,2 glycosyltransferase provided herein. In some furtherembodiments of the methods, at least one engineered glycosyltransferaseis a beta-1,3 glycosyltransferase provided herein. In yet someadditional embodiments of the methods, at least one engineeredglycosyltransferase is a beta-1,2 glycosyltransferase provided hereinand at least one additional engineered glycosyltransferase is a beta-1,3glycosyltransferase provided herein. In some further embodiments of themethods, at least one engineered sucrose synthase provided herein findsuse.

In the methods provided herein, at least one engineeredglycosyltransferase comprising a polypeptide sequence that has at least90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequenceidentity to SEQ ID NO: 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30,22, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66,68, 70, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104,106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132,134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160,162, 164, 166, 168, 170, 172, 174, 176, 178, 180, 182, 184, 186, 188,190, 192, 194, 196, 198, 200, 202, 204, 206, 208, 210, 212, 214, 216,218, 220, 222, 224, 226, 228, 230, 232, 234, 236, 238, 240, 242, 244,246, 248, 250, 252, 254, 256, 258, 260, 262, 264, 266, 268, 270, 272,274, 276, 278, 280, 282, 284, 286, 288, 290, 292, 294, 296, 298, 300,302, 304, 306, 308, 310, 312, 314, 316, 318, 320, 322, 324, 326, 328,330, 332, 334, 336, 338, 340, 342, 344, 346, 348, 350, 352, 354, 356,358, 360, 362, 364, 366, 368, 370, 372, 374, 376, 378, 380, 382, 384,386, 388, 390, 392, 394, 396, 398, 400, 402, 404, 406, 408, 410, 412,414, 416, 418, 420, 422, 424, 426, 428, 430, 432, 434, 436, 438, 440,442, 444, 446, 448, 450, 452, 454, 456, 458, 460, 462, 464, 466, 468,470, 472, 474, 476, 478, 480, 482, 484, 486, 488, 490, 492, 494, 496,498, 500, 502, 504, 506, 508, 510, 512, 514, 516, 518, 520, 522, 524,526, 528, 530, 532, 534, 536, 538, 540, 542, 544, 546, 548, 550, 552,554, 556, 558, 560, 562, 564, 566, 568, 570, 572, 574, 576, 578, 580,582, 584, 586, 588, 590, 592, 594, 596, 598, 600, 602, 604, 606, 608,610, 612, 614, 616, 618, 620, 622, 624, 626, 628, 630, 632, 634, 636,638, 640, 642, 644, 646, 648, 650, 652, 654, 656, 658, 660, 662, 664,666, 668, 670, 672, 674, 676, 678, 680, 682, 684, 686, 688, 690, 692,694, 696, 698, 700, 702, 704, 706, 708, 710, 712, 714, 716, 718, 720,722, 724, 726, 728, 730, 732, 734, 736, 738, 740, 742, 744, 746, 748,750, 752, 754, 770, 772, 774, 776, 778, 780, 782, 784, 786, 788, 790,792, 794, 796, 798, 800, 802, 804, 806, 808, 810, 812, 814, 816, 818,820, 822, 824, 826, 828, 830, 832, 834, 836, 838, 840, 842, 844, 846,848, 850, 852, 854, 856, 858, 860, 862, 864, 866, 868, 870, 872, 874,876, 878, 880, 882, 884, 886, 888, 890, 892, 894, 896, 898, 900, 902,904, 906, 908, 910, 912, 914, 916, 918, 920, 922, 924, 926, 928, 930,932, 934, 936, 938, 940, 942, 944, 946, 948, 950, 952, 954, 956, 958,960, 962, 964, 966, 968, 970, 972, 974, 976, 978, 980, 982, 984, 986,988, 990, 992, 994, 996, 998, 1000, 1002, 1004, 1006, 1008, 1010, 1012,1014, 1016, 1018, 1020, 1022, 1024, 1026, 1028, 1030, 1032, 1034, 1036,1038, 1040, 1042, 1044, 1046, 1048, 1050, 1052, 1054, 1056, 1058, 1060,1062, 1064, 1066, 1068, 1070, 1072, 1074, 1076, 1078, 1290, 1292, 1294,2596, 2598, 2600, 2602, 2604, 2606, 2608, 2610, 2612, 2614, 2616, 2618,2620, 2622, 2624, 2626, 2628, 2630, 2632, 2634, 2636, 2638, 2640, 2642,2644, 2646, 2648, 2650, 2652, 2654, 2656, 2658, 2660, 2662, 2664, 2666,2668, 2670, 2672, 2674, 2676, 2678, 2680, 2682, 2684, 2686, 2688, 2690,2692, 2694, 2696, 2698, 2700, 2702, 2704, 2706, 2708, 2710, 2712, 2714,2716, 2718, 2720, 2722, 2724, 2726, 2728, 2730, 2732, 2734, 2736, 2738,2740, 2742, 2744, 2746, 2748, 2750, 2752, 2754, 2756, 2758, 2760, 2762,2764, 2766, 2768, 2770, 2772, 2774, 2776, 2778, 2780, 2782, 2784, 2786,2788, 2790, 2792, 2794, 2796, 2798, 2800, 2802, 2804, 2806, 2808, 2810,2812, 2814, 2816, 2818, 2820, 2822, 2824, 2826, 2828, 2830, 2832, 2834,2836, 2838, 2840, 2842, 2844, 2846, 2848, 2850, 2852, 2854, 2856, 2858,2860, 2862, 2864, 2866, 2868, 2870, 2872, 2874, 2876, 2878, 2880, 2882,2884, 2886, 2888, 2890, 2892, 2894, 2896, 2898, 2900, 2902, 2904, 2906,2908, 2910, 2912, 2914, 2916, 2918, 2920, 2922, 2924, 2926, 2928, 2830,2932, 2934, 2936, 2938, 2940, 2942, 2944, 2946, 2948, 2950, 2952, 2954,2956, 2958, 2960, 2962, 2964, 2966, 2968, 2970, 2972, 2974, 2976, 2978,2980, 2982, 2984, 2986, 2988, 2990, 2992, 2994, 2996, 2998, 3000, 3002,3004, 3006, 3008, 3010, 3012, 3014, 3016, 3018, 3020, 3022, 3024, 3026,3028, 3030, 3032, 3034, 3036, 3038, 3040, 3042, 3044, 3046, 3048, 3050,3052, 3054, 3056, 3058, 3060, 3062, 3064, 3066, 3068, 3070, 3072, 3074,3076, 3078, 3080, 3082, 3084, 3086, 3088, 3090, 3092, 3094, 3096, 3098,3100, 3102, 3104, 3106, 3108, 3110, 3112, 3114, 3116, 3118, 3120, 3122,3124, 3126, 3128, 3130, 3132, 3134, 3136, 3138, 3140, 3142, 3144, 3146,3148, 3150, 3152, 3154, 3156, 3158, 3160, 3162, 3164, 3166, 3168, 3170,3172, 3174, 3176, 3178, 3180, 3182, 3184, 3186, 3188, 3190, 3192, 3194,3196, 3198, 3200, 3202, 3204, 3206, 3208, 3210, 3212, 3214, 3216, 3218,3220, 3222, 3224, 3226, 3228, 3230, 3232, 3234, 3236, 3238, 3240, 3242,3244, 3246, 3248, 3250, 3252, 3254, 3256, 3258, 3260, 3262, 3264, 3266,3268, 3270, 3272, 3274, 3276, 3278, 3280, 3282, 3284, 3286, 3288, 3290,3292, 3294, 3296, 3298, 3300, 3302, 3304, 3306, 3308, 3310, 3312, 3314,3316, 3318, 3320, 3322, 3324, 3326, 3328, 3330, 3332, 3334, 3336, 3338,3340, 3342, 3344, 3346, 3348, 3350, 3352, 3354, 3356, 3358, 3360, 3362,3364, 3366, 3368, 3370, 3372, 3374, 3376, 3378, 3380, 3382, 3384, 3386,3388, 3390, 3392, 3394, 3396, 3398, 3400, 3402, 3404, 3406, 3408, 3410,3412, 3414, 3416, 3418, 3420, 3422, 3424, 3426, 3428, 3430, 3432, 3434,3436, 3438, 3440, 3442, 3444, 3446, 3448, 3450, 3452, 3454, 3456, 3458,3460, 3462, 3464, 3466, 3468, 3470, 3472, 3474, 3476, 3478, 3480, 3482,3484, 3486, 3488, 3490, 3492, 3494, 3496, 3498, 3500, 3502, 3504, 3506,3508, 3510, 3512, 3514, 3516, 3518, 3520, 3522, 3524, 3526, 3528, 3530,3532, 3534, 3536, 3538, 3540, 3542, 3544, 3546, 3548, 3550, 3552, 3554,3556, 3558, 3560, 3562, 3564, 3566, 3568, 3570, 3572, 3574, 3576, 3578,3580, 3582, 3584, 3586, 3588, 3590, 3592, 3594, 3596, 3598, 3600, 3602,3604, 3606, 3608, 3610, 3612, 3614, 3616, 3618, 3620, 3622, 3624, 3626,3628, 3630, 3632, 3634, 3636, 3638, 3640, 3642, 3644, 3646, 3648, 3650,3652, 3654, 3656, 3658, 3660, 3662, 3664, 3666, 3668, 3670, 3672, 3674,3676, 3678, 3680, 3682, 3684, 3686, 3688, 3690, 3692, 3694, 3696, 3698,3700, 3702, 3704, 3706, 3708, 3710, 3712, 3714, 3716, 3718, 3720, 3722,3724, 3726, 3728, 3730, 3732, 3734, 3736, 3738, 3740, 3742, 3744, 3746,3748, 3750, 3752, 3754, 3756, 3758, 3760, 3762, 3764, 3766, 3768, 3770,3772, 3774, 3776, 3778, 3780, 3782, 3784, 3786, 3788, 3790, 3792, 3794,3796, 3798, 3800, 3802, 3804, 3806, 3808, 3810, 3812, 3814, 3816, 3818,3820, 3822, 3824, 3826, 3828, 3830, 3832, 3834, 3836, 3838, 3840, 3842,3844, 3846, 3848, 3850, 3852, 3854, 3856, 3858, 3860, 3862, 3864, 3866,3868, 3870, 3872, 3874, 3876, 3878, 3880, 3882, 3884, 3886, 3888, 3890,3892, 3894, 3896, 3898, 3900, 3902, 3904, 3906, 3908, 3910, 3912, 3914,3916, 3918, 3920, 3922, 3924, 3926, 3928, 3930, 3932, 3934, 3936, 3938,3940, 3942, 3944, 3946, 3948, 3950, 3952, 3954, 3956, 3958, 3960, 3962,3964, 3966, 3968, 3970, 3972, 3974, 3976, 3978, 3980, 3982, 3984, 3986,3988, 3990, 3992, 3994, 3996, 3998, 4000, 4002, 4004, 4006, 4008, 4010,4012, 4014, 4016, 4018, 4020, 4022, 4024, 4026, 4028, 4030, 4032, 4034,4036, 4038, 4040, 4042, 4044, 4046, 4048, 4050, 4052, 4054, 4056, 4058,4060, 4062, 4064, 4066, 4068, 4070, 4072, 4074, 4076, 4078, 4080, 4082,4084, 4086, 4088, 4090, 4092, 4094, 4096, 4098, 4100, 4102, 4104, 4106,4108, 4110, 4112, 4114, 4116, 4118, 4120, 4122, 4124, 4126, 4128, 4130,4132, 4134, 4136, 4138, 4140, 4142, 4144, 4146, 4148, 4150, 4152, 4154,4156, 4158, 4160, 4162, 4164, 4166, 4168, 4170, 4172, 4174, 4176, 4178,4180, 4182, 4184, 4186, 4188, 4190, 4192, 4194, 4196, 4198, 4200, 4202,4204, 4206, 4208, 4210, 4212, 4214, 4216, 4218, 4220, 4222, 4224, 4226,4228, 4230, 4232, 4234, 4236, 4238, 4240, 4242, 4244, 4246, 4248, 4250,4252, 4254, 4256, 4258, 4260, 4262, 4264, 4266, 4268, 4270, 4272, 4274,4276, 4278, 4280, 4282, 4284, 4286, 4288, 4290, 4292, 4294, 4296, 4298,4300, 4302, 4304, 4306, 4308, 4310, 4312, 4314, 4316, 4318, 4320, 4322,4324, 4326, 4328, 4330, 4332, 4334, 4336, 4338, 4340, 4342, 4344, 4346,4348, 4350, 4352, 4354, 4356, 4358, 4360, 4362, 4364, 4366, 4368, 4370,4372, 4374, 4376, 4378, 4380, 4382, 4384, 4386, 4388, 4390, 4392, 4394,4396, 4398, 4400, 4402, 4404, 4406, 4408, 4410, 4412, 4414, 4416, 4418,4420, 4422, 4424, 4426, 4428, 4430, 4432, 4434, 4436, 4438, 4440, 4442,4444, 4446, 4448, 4450, 4452, 4454, 4456, 4458, 4460, 4462, 4464, 4466,4468, 4470, 4472, 4474, 4476, 4478, 4480, 4482, 4484, 4486, 4488, 4490,4492, 4494, 4496, 4498, 4500, 4502, 4504, 4506, 4508, 4510, 4512, 4514,4516, 4518, 4520, 4522, 4524, 4526, 4528, 4530, 4532, 4534, 4536, 4538,4540, 4542, 4544, 4546, 4548, 4550, 4552, 4554, 4556, 4558, 4560, 4562,4564, 4566, 4568, 4570, 4572, 4574, 4576, 4578, 4580, 4582, 4584, 4586,4588, 4590, 4592, 4594, 4596, 4598, 4600, 4602, 4604, 4606, 4608, 4610,4612, 4614, 4616, 4618, 4620, 4622, 4624, 4626, 4628, 4630, 4632, 4634,4636, 4638, 4640, 4642, 4644, 4646, 4648, 4650, 4652, 4654, 4656, 4658,4660, 4662, 4664, 4666, 4668, 4670, 4672, 4674, 4676, 4678, 4680, 4682,4684, 4686, 4688, 4690, 4692, 4694, 4696, 4698, 4700, 4702, 4704, 4706,4708, 4710, 4712, 4714, 4716, 4718, 4720, 4722, 4724, 4726, 4728, 4730,4732, 4734, 4736, 4738, 4740, 4742, 4744, 4746, 4748, 4750, 4752, 4754,4756, 4758, 4760, 4762, 4764, 4766, 4768, 4770, 4772, 4774, 4776, 4778,4780, 4782, 4784, 4786, 4788, 4790, 4792, 4794, 4796, 4798, 4800, 4902,4804, 4806, 4808, 4810, 4812, 4814, 4816, 4818, 4820, 4822, 4824, 4826,4828, 4830, 4832, 4834, 4836, 4838, 4840, 4842, 4844, 4846, 4848, 4850,4852, 4854, 4856, 4858, 4860, 4862, 4864, 4866, 4868, 4870, 4872, 4874,4876, 4878, 4880, 4882, 4884, 4886, 4888, 4890, 4892, 4894, 4896, 4898,4900, 4902, 4904, 4906, 4908, 4910, 4912, 4914, 4916, 4918, 4920, 4922,4924, 4926, 4928, 4930, 4932, 4934, 4936, 4938, 4940, 4942, 4944, 4946,4948, 4950, 4952, 4954, 4956, 4958, 4960, 4962, 4964, 4966, 4968, 4970,4972, 4974, 4976, 4978, 4980, 4982, 4984, 4986, 4988, 4990, 4992, 4994,4996, 4998, 5000, 5002, 5004, 5006, 5008, 5010, 5012, 5014, 5016, 5018,5020, 5022, 5024, 5026, 5028, 5030, 5032, 5034, 5036, 5038, 5040, 5042,5044, 5046, 5048, 5050, 5052, 5054, 5056, 5058, 5060, 5062, 5064, 5066,5068, 5070, 5072, 5074, 5076, 5078, 5080, 5082, 5084, 5086, 5088, 5090,5092, 5094, 5096, 5098, 5100, 5102, 5104, 5106, 5108, 5110, 5112, 5114,5116, 5118, 5120, 5122, 5124, 5126, 5128, 5130, 5132, 5134, 5136, 5138,5140, 5142, 5144, 5146, 5148, 5150, 5152, 5154, 5156, 5158, 5160, 5162,5164, 5166, 5168, 5170, 5172, 5174, 5176, 5178, 5180, 5182, 5184, 5186,5188, 5190, 5192, 5194, 5196, 5198, 5200, 5202, 5204, 5206, 5208, 5210,5212, 5214, 5216, 5218, 5220, 5222, 5224, 5226, 5228, 5230, 5232, 5234,5236, 5238, 5240, 5242, 5244, 5246, 5248, 5250, 5252, 5254, 5256, 5258,5260, 5262, 5264, 5266, 5268, 5270, 5272, 5274, 5276, 5278, 5280, 5282,5284, 5286, 5288, 5290, 5292, 5294, 5296, 5298, 5300, 5302, 5304, 5306,5308, 5310, 5312, 5314, 5316, 5318, 5320, 5322, 5324, 5326, 5328, 5330,5332, 5334, 5336, 5338, 5340, 5342, 5344, 5346, 5348, 5350, 5352, 5354,5356, 5358, 5360, 5362, 5364, 5366, 5368, 5370, 5372, 5374, 5376, 5378,5380, 5382, 5384, 5386, 5388, 5390, 5392, 5394, 5396, 5398, 5400, 5402,5404, 5406, 5408, 5410, 5412, 5414, 5416, 5418, 5420, 5422, 5424, 5426,5428, 5430, 5432, 5434, 5436, 5438, 5440, 5442, 5444, 5446, 5448, 5450,5452, 5454, 5456, 5458, 5460, 5462, 5464, 5466, 5468, 5470, 5472, 5474,5476, 5478, 5480, 5482, 5484, 5486, 5488, 5490, 5492, 5494, 5496, 5498,5500, 5502, 5504, 5506, 5508, 5510, 5512, 5514, 5516, 5518, 5520, 5522,5524, 5526, 5528, 5530, 5532, 5534, 5536, 5538, 5540, 5542, 5544, 5546,5548, 5550, 5552, 5554, 5556, 5558, 5560, 5562, 5564, 5566, 5568, 5570,5572, 5574, 5576, 5578, 5580, 5582, 5584, 5586, 5588, 5590, 5592, 5594,5596, 5598, 5600, 5602, 5604, 5606, 5608, 5610, 5612, 5614, 5616, 5618,5620, 5622, 5624, 5626, 5628, 5630, 5632, 5634, 5636, 5638, 5640, 5642,5644, 5646, 5648, 5650, 5652, 5654, 5656, 5658, 5660, 5662, 5664, 5666,5668, 5670, 5672, 5674, 5676, 5678, 5680, 5682, 5684, 5686, 5688, 5690,5692, 5694, 5696, 5698, 5700, 5702, 5704, 5706, 5708, 5710, 5712, 5714,5716, 5718, 5720, 5722, 5724, 5726, 5728, 5730, 5732, 5734, 5736, 5738,5740, 5742, 5744, 5746, 5748, 5750, 5752, 5754, 5756, 5758, 5760, 5762,5764, 5766, 5768, 5770, 5772, 5774, 5776, 5778, 5780, 5782, 5784, 5786,5788, 5790, 5792, 5794, 5796, 5798, 5800, 5802, 5804, 5806, 5808, 5810,5812, 5814, 5816, 5818, 5820, 5822, 5824, 5826, 5828, 5830, 5832, 5834,5836, 5838, 5840, 5842, 5844, 5846, 5848, 5850, 5852, 5854, 5856, 5858,5860, 5862, 5864, 5866, 5868, 5870, 5872, 5874, 5876, 5878, 5880, 5882,5884, 5886, 5888, 5890, 5892, 5894, 5896, 5898, 5900, 5902, 5904, 5906,5908, 5910, 5912, 5914, 5916, 5918, 5920, 5922, 5924, 5926, 5928, 5930,5932, 5934, 5936, 5938, 5940, 5942, 5944, 5946, 5948, 5950, 5952, 5954,5956, 5958, 5960, 5962, 5964, 5966, 5968, 5970, 5972, 5974, 5976, 5978,5980, 5982, 5984, 5986, 5988, 5990, 5992, 5994, 5996, 5998, 6000, 6002,6004, 6006, 6008, 6010, 6012, 6014, 6016, 6018, 6020, 6022, 6024, 6026,6028, 6030, 6032, 6034, 6036, 6038, 6040, 6042, 6044, 6046, 6048, 6050,6052, 6054, 6056, 6058, 6060, 6062, 6064, 6066, 6068, 6070, 6072, 6074,6076, 6078, 6080, 6082, 6084, 6086, 6088, 6090, 6092, 6094, 6096, 6098,6100, 6102, 6104, 6106, 6108, 6110, 6112, 6114, 6116, 6118, 6120, 6122,6124, 6126, 6128, 6130, 6132, 6134, 6136, 6138, 6140, 6142, 6144, 6146,6148, 6150, 6152, 6154, 6156, 6158, 6160, 6162, 6164, 6166, 6168, 6170,6172, 6174, 6176, 6178, 6180, 6182, 6184, 6186, 6188, 6190, 6192, 6194,6196, 6198, 6200, 6202, 6204, 6206, 6208, 6210, 6212, 6214, 6216, 6218,6220, 6222, 6224, 6226, 6228, 6230, 6232, 6234, 6236, 6238, 6240, 6242,6244, 6246, 6248, 6250, 6252, 6254, 6256, 6258, 6260, 6262, 6264, 6266,6268, 6270, 6272, 6274, 6276, 6278, 6280, 6282, 6284, 6286, 6288, 6290,6292, 6294, 6296, 6298, 6300, 6302, 6304, 6306, 6308, 6310, 6312, 6314,6316, 6318, 6320, 6322, 6324, 6326, 6328, 6330, 6332, 6334, 6336, 6338,6340, 6342, 6344, 6346, 6348, 6350, 6352, 6354, 6356, 6358, 6360, 6362,6364, 6366, 6368, 6370, 6372, 6374, 6376, 6378, 6380, 6382, 6384, 6386,6388, 6390, 6392, 6394, 6396, 6398, 6400, 6402, 6404, 6406, 6408, 6410,6412, 6414, 6416, 6418, 6420, 6422, 6424, 6426, 6428, 6430, 6432, 6434,6436, 6438, 6440, 6442, 6444, 6446, 6448, 6450, 6452, 6454, 6456, 6458,6460, 6462, 6464, 6466, 6468, 6470, 6472, 6474, 6476, 6478, 6480, 6482,6484, 6486, 6488, 6490, 6492, 6494, 6496, 6498, 6500, 6502, 6504, 6506,6508, 6510, 6512, 6514, 6516, 6518, 6520, 6522, 6524, 6526, 6528, 6530,6532, 6534, 6536, 6538, 6540, 6542, 6544, 6546, 6548, 6550, 6552, 6554,6556, 6558, 6560, 6562, 6564, 6566, 6568, 6570, 6572, 6574, 6576, 6578,6580, 6582, 6584, 6586, 6588, 6590, 6592, 6594, 6596, 6598, 6600, 6602,6604, 6606, 6608, 6610, 6612, 6614, 6616, 6618, 6620, 6622, 6624, 6626,6628, 6630, 6632, 6634, 6636, 6638, 6640, 6642, 6644, 6646, 6648, 6650,6652, 6654, 6656, 6658, 6660, 6662, 6664, 6666, 6668, 6670, 6672, 6674,6676, 6678, 6680, 6682, 6684, 6686, 6688, 6690, 6692, 6694, 6696, 6698,6700, 6702, 6704, 6706, 6708, 6710, 6712, 6714, 6716, 6718, 6720, 6722,6724, 6726, 6728, 6730, 6732, 6734, 6736, 6738, 6740, 6742, 6744, 6746,6748, 6750, 6752, 6754, 6756, 6758, 6760, 6762, 6764, 6766, 6768, 6770,6772, 6774, 6776, 6778, 6780, 6782, 6784, 6786, 6788, 6790, 6792, 6794,6796, 6798, 6800, 6802, 6804, 6806, 6808, 6810, 6812, 6814, 6816, 6818,6820, 6822, 6824, 6826, 6828, 6830, 6832, 6834, 6836, 6838, 6840, 6842,6844, 6846, 6848, 6850, 6852, 6854, 6856, 6858, 6860, 6862, 6864, 6866,6868, 6870, 6872, 6874, 6876, 6878, 6880, 6882, 6884, 6886, 6888, 6890,6892, 6894, 6896, 6898, 6900, 6902, 6904, 6906, 6908, 6910, 6912, 6914,6916, 6918, 6920, 6922, 6924, 6926, 6928, 6930, 6932, 6934, 6936, 6938,6940, 6942, 6944, 6946, 6948, 6950, 6952, 6954, 6956, 6958, 6960, 6962,6964, 6966, 6968, 6970, 6972, 6974, 6976, 6978, 6980, 6982, 6984, 6986,6988, 6990, 6992, 6994, 6996, 6998, 7000, 7002, 7004, 7006, 7008, 7010,7012, 7014, 7016, 7018, 7020, 7022, 7024, 7026, 7028, 7030, 7032, 7034,7036, 7038, 7040, 7042, 7044, 7046, 7048, 7050, 7052, 7054, 7056, 7058,7060, 7062, 7064, 7066, 7068, 7070, 7072, 7074, 7076, 7078, 7080, 7082,7084, 7086, 7088, 7090, 7092, 7094, 7096, 7098, 7100, 7102, 7104, 7106,7108, 7110, 7112, 7114, 7116, 7118, 7120, 7122, 7124, 7126, 7128, 7130,7132, 7134, 7136, 7138, 7140, 7142, 7144, 7146, 7148, 7150, 7152, 7154,7156, 7158, 7160, 7162, 7164, 7166, 7168, 7170, 7172, 7174, 7176, 7178,7180, 7182, 7184, 7186, 7188, 7190, 7192, 7194, 7196, 7198, 7200, 7202,7204, 7206, 7208, 7210, 7212, 7214, 7216, 7218, 7220, 7222, 7224, 7226,7228, 7230, 7232, 7234, 7236, 7238, 7240, 7242, 7244, 7246, 7248, 7250,7252, 7254, 7256, 7258, 7260, 7262, 7264, 7266, 7268, 7270, 7272, 7274,7276, 7278, 7280, 7282, 7284, 7286, 7288, 7290, 7292, 7294, 7296, 7298,7300, 7302, 7304, 7306, 7308, 7310, 7312, 7314, 7316, 7318, 7320, 7322,7324, 7326, 7328, 7330, 7332, 7334, 7336, 7338, 7340, 7342, 7344, 7346,7348, 7350, 7352, 7354, 7356, 7358, 7360, 7362, 7364, 7366, 7368, 7370,7372, 7374, 7376, 7378, 7380, 7382, 7384, 7386, 7388, 7390, 7392, 7394,7396, 7398, 7400, 7402, 7404, 7406, 7408, 7410, 7412, 7414, 7416, 7418,7420, 7422, 7424, 7426, 7428, 7430, 7432, 7434, 7436, 7766, 7768, 7770,7772, 7774, 7776, 7778, 7780, 7782, 7784, 7786, 7788, 7790, 7792, 7794,7796, 7798, 7800, 7812, 7814, 7816, 7818, 7820, 7822, 7824, 7826, 7828,7830, 7832, 7834, 7836, 7838, 7840, 7842, 7844, 7846, 7848, 7850, 7852,7854, 7856, 7858, 7860, 7862, 7864, 7866, 7868, 7870, 7872, 7874, 7876,7878, 7880, 7882, 7884, 7886, 7888, 7890, 7892, 7894, 7896, 7898, 7900,7902, 7904, 7906, 7908, 7910, 7912, 7914, 7916, 7918, 7920, 7922, 7924,7926, 7928, 7930, 7932, 7934, 7936, 7938, 7940, 7942, 7944, 7946, 7948,7950, 7952, 7954, 7956, 7958, 7960, 7962, 7964, 7966, 7968, 7970, 7972,7974, 7976, 7978, 7980, 7982, 7984, 7986, 7988, 7990, 7992, 7994, 7996,7998, 8000, 8002, 8004, 8006, 8008, 8010, 8012, 8014, 8016, 8018, 8020,8022, 8024, 8026, 8028, 8030, 8032, 8034, 8036, 8038, 8040, 8042, 8044,8046, 8048, 8050, 8052, 8054, 8056, 8058, 8060, 8062, 8064, 8066, 8068,8070, 8072, 8074, 8076, 8078, 8080, 8082, 8084, 8086, 8088, 8090, 8092,8094, 8096, 8098, 8100, 8102, 8104, 8106, 8108, 8110, 8112, 8114, 8116,8118, 8120, 8122, 8124, 8126, 8128, 8130, 8132, 8134, 8136, 8138, 8140,8142, 8144, 8146, 8148, 8150, 8152, 8154, 8156, 8158, 8160, 8162, 8164,8166, 8168, 8170, 8172, 8174, 8176, 8178, 8180, 8182, 8184, 8186, 8188,8190, 8192, 8194, 8196, 8198, 8200, 8202, 8204, 8206, 8208, 8210, 8212,8214, 8216, 8218, 8220, 8222, 8224, 8226, 8228, 8230, 8232, 8234, 8236,8238, 8240, 8242, 8244, 8246, 8248, 8250, 8252, 8254, 8256, 8258, 8260,8262, 8264, 8266, 8268, 8270, 8272, 8274, 8276, 8278, 8280, 8282, 8284,8286, 8288, 8290, 8292, 8294, 8296, 8298, 8300, 8302, 8304, 8306, 8308,8310, 8312, 8314, 8316, 8318, 8320, 8322, 8324, 8326, 8328, 8330, 8332,8334, 8336, 8338, 8340, 8342, 8344, 8346, 8348, 8350, 8352, 8354, 8356,8358, 8360, 8362, 8364, 8366, 8368, 8482, 8484, 8486, 8488, 8490, 8492,8494, 8496, 8498, 8500, 8502, 8504, 8506, 8508, 8510, 8512, 8514, 8516,8518, 8520, 8522, 8524, 8526, 8528, 8530, 8532, 8534, 8536, 8538, 8540,8542, 8544, 8546, 8548, 8550, 8552, 8554, 8556, 8558, 8560, 8562, 8564,8566, 8568, 8570, 8572, 8574, 8576, 8578, 8580, 8582, 8584, 8586, 8588,8590, 8592, 8594, 8596, 8598, 8600, 8602, 8604, 8606, 8608, 8610, 8612,8614, 8616, 8618, 8620, 8622, 8624, 8626, 8628, 8630, 8632, 8634, 8636,8638, 8640, 8642, 8644, 8646, 8648, 8650, 8652, 8654, 8656, 8658, 8660,8662, 8664, 8666, 8668, 8670, 8672, 8674, 8676, 8678, 8680, 8682, 8684,8686, 8688, 8690, 8692, 8694, 8696, 8698, 8700, 8702, 8704, 8706, 8708,8710, 8712, 8714, 8716, 8718, 8720, 8722, 8724, 8726, 8728, 8730, 8732,8734, 8736, 8738, 8740, 8742, 8744, 8746, 8748, 8750, 8752, 8754, 8756,8758, 8760, 8762, 8764, 8766, 8768, 8770, 8772, 8774, 8776, 8778, 8780,8782, 8784, 8786, 8788, 8790, 8792, 8794, 8796, 9108, 9110, 9112, 9114,9116, 9118, 9120, 9122, 9124, 9126, 9128, 9130, 9132, 9134, 9136, 9138,9140, 9142, 9144, 9146, 9148, 9150, 9152, 9154, 9156, 9158, 9160, 9162,9164, 9166, 9168, 9170, 9172, 9174, 9176, 9178, 9180, 9182, 9184, 9186,9188, 9190, 9192, 9194, 9196, 9198, 9200, 9202, 9204, 9206, 9208, 9210,9212, 9214, 9216, 9218, 9220, 9222, 9224, 9226, 9228, 9230, 9232, 9234,9236, 9238, and/or 9240 finds use.

In the methods provided herein, at least one engineeredglycosyltransferase comprising polypeptide sequence that has at least90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequenceidentity to SEQ ID NO: 1080, 1082, 1084, 1086, 1088, 1090, 1092, 1094,1096, 1098, 1100, 1102, 1104, 1106, 1108, 1110, 1112, 1114, 1116, 1118,1120, 1122, 1124, 1126, 1128, 1130, 1132, 1134, 1136, 1138, 1140, 1142,1144, 1146, 1148, 1150, 1152, 1154, 1156, 1158, 1160, 1162, 1164, 1166,1168, 1170, 1172, 1174, 1176, 1178, 1180, 1182, 1184, 1186, 1188, 1190,1192, 1194, 1196, 1198, 1200, 1202, 1204, 1206, 1208, 1210, 1212, 1214,1216, 1218, 1220, 1222, 1224, 1226, 1228, 1230, 1232, 1234, 1236, 1238,1240, 1242, 1244, 1246, 1248, 1250, 1252, 1254, 1256, 1258, 1260, 1262,1264, 1266, 1268, 1270, 1272, 1274, 1276, 1278, 1280, 1282, 1284, 1286,1288, 1296, 1298, 1300, 1302, 1304, 1306, 1308, 1310, 1312, 1314, 1316,1318, 1320, 1322, 1324, 1326, 1328, 1330, 1332, 1334, 1336, 1338, 1340,1342, 1344, 1346, 1348, 1350, 1352, 1354, 1356, 1358, 1360, 1362, 1364,1366, 1368, 1370, 1372, 1374, 1376, 1378, 1380, 1382, 1384, 1386, 1388,1390, 1392, 1394, 1396, 1398, 1400, 1402, 1404, 1406, 1408, 1410, 1412,1414, 1416, 1418, 1420, 1422, 1424, 1426, 1428, 1430, 1432, 1434, 1436,1438, 1440, 1442, 1444, 1446, 1448, 1450, 1452, 1454, 1456, 1458, 1460,1462, 1464, 1466, 1468, 1470, 1472, 1474, 1476, 1478, 1480, 1482, 1484,1486, 1488, 1490, 1492, 1494, 1496, 1498, 1500, 1502, 1504, 1506, 1508,1510, 1512, 1514, 1516, 1518, 1520, 1522, 1524, 1526, 1528, 1530, 1532,1534, 1536, 1538, 1540, 1542, 1544, 1546, 1548, 1550, 1552, 1554, 1556,1558, 1560, 1562, 1564, 1566, 1568, 1570, 1572, 1574, 1576, 1578, 1580,1582, 1584, 1586, 1588, 1590, 1592, 1594, 1596, 1598, 1600, 1602, 1604,1606, 1608, 1610, 1612, 1614, 1616, 1618, 1620, 1622, 1624, 1626, 1628,1630, 1632, 1634, 1636, 1638, 1640, 1642, 1644, 1646, 1648, 1650, 1652,1654, 1656, 1658, 1660, 1662, 1664, 1666, 1668, 1670, 1672, 1674, 1676,1678, 1680, 1682, 1684, 1686, 1688, 1690, 1692, 1694, 1696, 1698, 1700,1702, 1704, 1706, 1708, 1710, 1712, 1714, 1716, 1718, 1720, 1722, 1724,1726, 1728, 1730, 1732, 1734, 1736, 1738, 1740, 1742, 1744, 1746, 1748,1750, 1752, 1754, 1756, 1758, 1760, 1762, 1764, 1766, 1768, 1770, 1772,1774, 1776, 1778, 1780, 1782, 1784, 1786, 1788, 1790, 1792, 1794, 1796,1798, 1800, 1802, 1804, 1806, 1808, 1810, 1812, 1814, 1816, 1818, 1820,1822, 1824, 1826, 1828, 1830, 1832, 1834, 1836, 1838, 1840, 1842, 1844,1846, 1848, 1850, 1852, 1854, 1856, 1858, 1860, 1862, 1864, 1866, 1868,1870, 1872, 1874, 1876, 1878, 1880, 1882, 1884, 1886, 1888, 1890, 1892,1894, 1896, 1898, 1900, 1902, 1904, 1906, 1908, 1910, 1912, 1914, 1916,1918, 1920, 1922, 1924, 1926, 1928, 1930, 1932, 1934, 1936, 1938, 1940,1942, 1944, 1946, 1948, 1950, 1952, 1954, 1956, 1958, 1960, 1962, 1964,1966, 1968, 1970, 1972, 1974, 1976, 1978, 1980, 1982, 1984, 1986, 1988,1990, 1992, 1994, 1996, 1998, 2000, 2002, 2004, 2006, 2008, 2010, 2012,2014, 2016, 2018, 2020, 2022, 2024, 2026, 2028, 2030, 2032, 2034, 2036,2038, 2040, 2042, 2044, 2046, 2048, 2050, 2052, 2054, 2056, 2058, 2060,2062, 2064, 2066, 2068, 2070, 2072, 2074, 2076, 2078, 2080, 2082, 2084,2086, 2088, 2090, 2092, 2094, 2096, 2098, 2100, 2102, 2104, 2106, 2108,2110, 2112, 2114, 2116, 2118, 2120, 2122, 2124, 2126, 2128, 2130, 2132,2134, 2136, 2138, 2140, 2142, 2144, 2146, 2148, 2150, 2152, 2154, 2156,2158, 2160, 2162, 2164, 2166, 2168, 2170, 2172, 2174, 2176, 2178, 2180,2182, 2184, 2186, 2188, 2190, 2192, 2194, 2196, 2198, 2200, 2202, 2204,2206, 2208, 2210, 2212, 2214, 2216, 2218, 2220, 2222, 2224, 2226, 2228,2230, 2232, 2234, 2236, 2238, 2240, 2242, 2244, 2246, 2248, 2250, 2252,2254, 2256, 2258, 2260, 2262, 2264, 2266, 2268, 2270, 2272, 2274, 2276,2278, 2280, 2282, 2284, 2286, 2288, 2290, 2292, 2294, 2296, 2298, 2300,2302, 2304, 2306, 2308, 2310, 2312, 2314, 2316, 2318, 2320, 2322, 2324,2326, 2328, 2330, 2332, 2334, 2336, 2338, 2340, 2342, 2344, 2346, 2348,2350, 2352, 2354, 2356, 2358, 2360, 2362, 2364, 2366, 2368, 2370, 2372,2374, 2376, 2378, 2380, 2382, 2384, 2386, 2388, 2390, 2392, 2394, 2396,2398, 2400, 2402, 2404, 2406, 2408, 2410, 2412, 2414, 2416, 2418, 2420,2422, 2424, 2426, 2428, 2430, 2432, 2434, 2436, 2438, 2440, 2442, 2444,2446, 2448, 2450, 2452, 2454, 2456, 2458, 2460, 2462, 2464, 2466, 2468,2470, 2472, 2474, 2476, 2478, 2480, 2482, 2484, 2486, 2488, 2490, 2492,2494, 2496, 2498, 2500, 2502, 2504, 2506, 2508, 2510, 2512, 2514, 2516,2518, 2520, 2522, 2524, 2526, 2528, 2530, 2532, 2534, 2536, 2538, 2540,2542, 2544, 2546, 2548, 2550, 2552, 2554, 2556, 2558, 2560, 2562, 2564,2566, 2568, 2570, 2572, 2574, 2576, 2578, 2580, 2582, 2584, 2586, 2588,2590, 2592, 2594, 7438, 7440, 7442, 7444, 7446, 7448, 7450, 7452, 7454,7456, 7458, 7460, 7462, 7464, 7466, 7468, 7470, 7472, 7474, 7476, 7478,7480, 7482, 7484, 7486, 7488, 7490, 7492, 7494, 7496, 7498, 7500, 7502,7504, 7506, 7508, 7510, 7512, 7514, 7516, 7518, 7520, 7522, 7524, 7526,7528, 7530, 7532, 7534, 7536, 7538, 7540, 7542, 7544, 7546, 7548, 7550,7552, 7554, 7556, 7558, 7560, 7562, 7564, 7566, 7568, 7570, 7572, 7574,7576, 7578, 7580, 7582, 7584, 7586, 7588, 7590, 7592, 7594, 7596, 7598,7600, 7602, 7604, 7606, 7608, 7610, 7612, 7614, 7616, 7618, 7620, 7622,7624, 7626, 7628, 7630, 7632, 7634, 7636, 7638, 7640, 7642, 7644, 7646,7648, 7650, 7652, 7654, 7656, 7658, 7660, 7662, 7664, 7666, 7668, 7670,7672, 7674, 7676, 7678, 7680, 7682, 7684, 7686, 7688, 7690, 7692, 7694,7696, 7698, 7700, 7702, 7704, 7706, 7708, 7710, 7712, 7714, 7716, 7718,7720, 7722, 7724, 7726, 7728, 7730, 7732, 7734, 7736, 7738, 7740, 7742,7744, 7746, 7748, 7750, 7752, 7754, 7756, 7758, 7760, 7762, 7764, 8370,8372, 8374, 8376, 8378, 8380, 8382, 8384, 8386, 8388, 8390, 8392, 8394,8396, 8398, 8400, 8402, 8404, 8406, 8408, 8410, 8412, 8414, 8416, 8418,8420, 8422, 8424, 8426, 8428, 8430, 8432, 8434, 8436, 8438, 8440, 8442,8444, 8446, 8448, 8450, 8452, 8454, 8456, 8458, 8460, 8462, 8464, 8466,8468, 8470, 8472, 8474, 8476, 8478, 8480, 8798, 8800, 8802, 8804, 8806,8808, 8810, 8812, 8814, 8816, 8818, 8820, 8822, 8824, 8826, 8828, 8830,8832, 8834, 8836, 8838, 8840, 8842, 8844, 8846, 8848, 8850, 8852, 8854,8856, 8858, 8860, 8862, 8864, 8866, 8868, 8870, 8872, 8874, 8876, 8878,8880, 8882, 8884, 8886, 8888, 8890, 8892, 8894, 8896, 8898, 8900, 8902,8904, 8906, 8908, 8910, 8912, 8914, 8916, 8918, 8920, 8922, 8924, 8926,8928, 8930, 8932, 8934, 8936, 8938, 8940, 8942, 8944, 8946, 8948, 8950,8952, 8954, 8956, 8958, 8960, 8962, 8964, 8966, 8968, 8970, 8972, 8974,8976, 8978, 8980, 8982, 8984, 8986, 8988, 8990, 8992, 8994, 8996, 8998,9000, 9002, 9004, 9006, 9008, 9010, 9012, 9014, 9016, 9018, 9020, 9022,9024, 9026, 9028, 9030, 9032, 9034, 9036, 9038, 9040, 9042, 9044, 9046,9048, 9050, 9052, 9054, 9056, 9058, 9060, 9062, 9064, 9066, 9068, 9070,9072, 9074, 9076, 9078, 9080, 9082, 9084, 9086, 9088, 9090, 9092, 9094,9096, 9098, 9100, 9102, 9104, and/or 9106 finds use.

The present invention also provides rebaudiosides and compositionscomprising the rebaudiosides produced according to the methods providedherein. In some embodiments, the rebaudioside is rebaudioside M, whilein some alternative embodiments, the rebaudioside is rebaudioside A, andin still further embodiments, the rebaudioside is rebaudioside I, and insome additional embodiments, the rebaudioside is rebaudioside D. In somefurther embodiments, the present invention provides a mixture ofrebaudiosides produced according to the methods provided herein,including mixtures of rebaudioside M, rebaudioside A, rebaudioside I,and/or rebaudioside D, in any combination of concentrations ofrebaudiosides and other components of interest. In some embodiments, thepresentation invention provides compositions comprising mixtures ofrebaudioside M, rebaudioside A, rebaudioside I, and/or rebaudioside D,in any combination of concentrations of rebaudiosides and othercomponents of interest. Indeed, it is not intended that the presentinvention be limited to any particular combination or mixture ofrebaudioside(s) produced according to the methods of the presentinvention.

The present invention provides methods for producing rebaudioside M,comprising providing a rebaudioside D and/or rebaudioside I substrate,NDP-glucose, and a least one engineered NDP-glycosyltransferase providedherein, combining the rebaudioside D and rebaudioside I substrate,NDP-glucose and an NDP-glycosyltransferase under conditions such thatrebaudioside M is produced.

The present invention also provides methods for producing rebaudiosideM, comprising providing a rebaudioside D substrate, NDP-glucose, and aleast one engineered NDP-glycosyltransferase provided herein, combiningthe rebaudioside D substrate, NDP-glucose and an NDP-glycosyltransferaseunder conditions such that rebaudioside M is produced.

The present invention also provides methods for producing rebaudiosideM, comprising providing a rebaudioside I substrate, NDP-glucose, and aleast one engineered NDP-glycosyltransferase provided herein, combiningthe rebaudioside I substrate, NDP-glucose and an NDP-glycosyltransferaseunder conditions such that rebaudioside M is produced.

The present invention also provides methods for producing rebaudioside Aand/or rebaudioside I, comprising providing a stevioside substrate,NDP-glucose, and at least one engineered NDP-glycosyltransferaseprovided herein, combining the stevioside substrate, NDP-glucose and anNDP-glycosyltransferase under conditions such that rebaudioside A and/orrebaudioside I is produced.

The present invention also provides methods for producing rebaudiosideA, comprising providing a stevioside substrate, NDP-glucose, and atleast one engineered NDP-glycosyltransferase provided herein, combiningthe stevioside substrate, NDP-glucose and an NDP-glycosyltransferaseunder conditions such that rebaudioside A is produced.

The present invention also provides methods for producing rebaudiosideI, comprising providing a stevioside substrate, NDP-glucose, and atleast one engineered NDP-glycosyltransferase provided herein, combiningthe stevioside substrate, NDP-glucose and an NDP-glycosyltransferaseunder conditions such that rebaudioside I is produced.

The present invention also provides methods for producing rebaudiosideD, comprising providing a stevioside substrate, NDP-glucose, and atleast one engineered NDP-glycosyltransferase provided herein, combiningthe stevioside substrate, NDP-glucose and an NDP-glycosyltransferaseunder conditions such that rebaudioside D is produced.

The present invention also provides methods for producing rebaudioside Mcomprising providing a rebaudioside D and/or rebaudioside I substrate,ADP-glucose, and at least one engineered ADP-glycosyltransferaseprovided herein, combining the rebaudioside D and/or rebaudioside Isubstrate, ADP-glucose and an ADP-glycosyltransferase under conditionssuch that rebaudioside M is produced.

The present invention also provides methods for producing rebaudioside Mcomprising providing a rebaudioside D substrate, ADP-glucose, and atleast one engineered ADP-glycosyltransferase provided herein, combiningthe rebaudioside D substrate, ADP-glucose and an ADP-glycosyltransferaseunder conditions such that rebaudioside M is produced.

The present invention also provides methods for producing rebaudioside Mcomprising providing a rebaudioside I substrate, ADP-glucose, and atleast one engineered ADP-glycosyltransferase provided herein, combiningthe rebaudioside I substrate, ADP-glucose and an ADP-glycosyltransferaseunder conditions such that rebaudioside M is produced.

The present invention also provides methods for producing rebaudioside Aand/or rebaudioside I, comprising providing a stevioside substrate,ADP-glucose, and at least one engineered ADP-glycosyltransferaseprovided herein, combining the stevioside substrate, ADP-glucose and anADP-glycosyltransferase under conditions such that rebaudioside A and/orrebaudioside I is produced.

The present invention also provides methods for producing rebaudiosideA, comprising providing a stevioside substrate, ADP-glucose, and atleast one engineered ADP-glycosyltransferase provided herein, combiningthe stevioside substrate, ADP-glucose and an ADP-glycosyltransferaseunder conditions such that rebaudioside A is produced.

The present invention also provides methods for producing rebaudiosideI, comprising providing a stevioside substrate, ADP-glucose, and atleast one engineered ADP-glycosyltransferase provided herein, combiningthe stevioside substrate, ADP-glucose and an ADP-glycosyltransferaseunder conditions such that rebaudioside I is produced.

The present invention also provides methods for producing rebaudiosideD, comprising providing a stevioside substrate, ADP-glucose, and atleast one engineered ADP-glycosyltransferase provided herein, and/or118, combining the stevioside substrate, ADP-glucose and anADP-glycosyltransferase under conditions such that rebaudioside D isproduced.

The present invention also provides methods for producing rebaudioside Mcomprising providing a rebaudioside D substrate, NDP, sucrose, a sucrosesynthase, and at least one engineered ADP-glycosyltransferase providedherein, combining the rebaudioside D substrate, NDP, sucrose, sucrosesynthase and an ADP-glycosyltransferase under conditions such thatrebaudioside M is produced. In some embodiments, the sucrose synthase isan engineered sucrose synthase provided herein. In some embodiments, asugar other than sucrose finds use, in combination with the appropriatesynthase.

The present invention also provides methods for producing rebaudioside Aand/or rebaudioside I comprising providing a stevioside substrate, NDP,sucrose, a sucrose synthase, and at least one engineeredADP-glycosyltransferase provided herein, combining the steviosidesubstrate, NDP, sucrose, sucrose synthase and an ADP-glycosyltransferaseunder conditions such that rebaudioside A and/or rebaudioside I isproduced. In some embodiments, the sucrose synthase is an engineeredsucrose synthase provided herein. In some embodiments, a sugar otherthan sucrose finds use, in combination with the appropriate synthase.

The present invention also provides methods for producing rebaudiosideA, comprising providing a stevioside substrate, NDP, sucrose, a sucrosesynthase, and at least one engineered ADP-glycosyltransferase providedherein, combining the stevioside substrate, NDP, sucrose, sucrosesynthase and an ADP-glycosyltransferase under conditions such thatrebaudioside A is produced. In some embodiments, the sucrose synthase isan engineered sucrose synthase provided herein. In some embodiments, asugar other than sucrose finds use, in combination with the appropriatesynthase.

The present invention also provides methods for producing rebaudiosideI, comprising providing a stevioside substrate, NDP, sucrose, a sucrosesynthase, and at least one engineered ADP-glycosyltransferase providedherein, combining the stevioside substrate, NDP, sucrose, sucrosesynthase and an ADP-glycosyltransferase under conditions such thatrebaudioside A and/or rebaudioside I is produced. In some embodiments,the sucrose synthase is an engineered sucrose synthase provided herein.In some embodiments, a sugar other than sucrose finds use, incombination with the appropriate synthase.

The present invention also provides methods for producing rebaudiosideD, comprising providing a stevioside substrate, NDP, sucrose, a sucrosesynthase, and at least one engineered ADP-glycosyltransferase providedherein, combining the stevioside substrate, NDP, sucrose, sucrosesynthase and an ADP-glycosyltransferase under conditions such thatrebaudioside D is produced. In some embodiments, sucrose synthase is anengineered sucrose synthase provided herein. In some embodiments, asugar other than sucrose finds use, in combination with the appropriatesynthase.

The present invention also provides methods for producing rebaudiosideM, comprising providing a stevioside substrate comprising at least onestevioside and/or a mixture of steviosides and rebA, NDP, sucrose, asucrose synthase, and at least one engineered ADP-glycosyltransferaseprovided herein, combining the stevioside substrate, NDP, sucrose,sucrose synthase and an ADP-glycosyltransferase under conditions suchthat rebaudioside D is produced. In some embodiments, the sucrosesynthase is an engineered sucrose synthase provided herein. In someembodiments, a sugar other than sucrose finds use, in combination withthe appropriate synthase.

The present invention also provides methods of producing rebaudioside M,comprising providing a stevioside substrate, NDP, sucrose, at least onesucrose synthase, and at least one engineered ADP-glycosyltransferaseprovided herein, combining the stevioside substrate, NDP, and anADP-glycosyltransferase under conditions such that rebaudioside A isfirst produced, rebaudioside D and/or rebaudioside I is then produced,and rebaudioside M finally produced. In some embodiments, the methodsfurther comprise sucrose and sucrose synthase. In some embodiments, thesucrose synthase is an engineered sucrose synthase provided herein.

In some embodiments, the methods provided by the present invention areconducted as one-pot reaction, while in some alternative embodiments,the methods are conducted in multiple reaction vessels. In someembodiments, the methods are conducted in single and/or multiplereaction vessels in a sequential manner. In some embodiments, the methodsteps are repeated (i.e., there are multiple iterations of some or allof the steps of the methods). In some embodiments, the sucrose isrecycled during repeated steps. In some additional embodiments,engineered glycosyltransferase and/or other reaction components (e.g.,co-factors) are recycled. In some embodiments of the methods, thestevioside substrate is extracted from Stevia rebaudiana, while in somealternative methods, the stevioside substrate is synthetically produced.In some further embodiments, the glycosyltransferase is immobilized. Insome additional embodiments, the sucrose synthase is immobilized. Insome further embodiments, the glycosyltransferase and/or the sucrosesynthase is immobilized. In some embodiments of the methods, fructose isproduced. In some additional embodiments, fructose is removed from thereaction products.

The present invention also provides compositions comprising at least oneengineered glycosyltransferase variant provided herein. The presentinvention also provides compositions comprising at least onenon-naturally occurring glycosyltransferase variant as provided herein.

The present invention also provides methods for glycosylation of asubstrate to produce a beta-glycosylated product, comprising the stepsof: providing at least one glycosyl group donor, a least one glycosylgroup acceptor, and at least one glycosyltransferase enzyme; contactingthe glycosyl group donor and glycosyl group acceptor with theglycosyltransferase enzyme under conditions such that the glycosyl groupacceptor is glycosylated to produce at least one product havingbeta-glucose linkages. In some embodiments of the methods, the glycosylgroup donor is a nucleotide diphosphate sugar, for example adeninediphosphoglucose (ADP-glucose). In some further embodiments of themethods, the glycosyl group acceptor is selected from glycosyl, alkoxy,carboxy, aminocarbonyl, heteroalkyl, heteroalkenyl, heteroalkynyl,carboxyalkyl, aminoalkyl, haloalkyl, alkylthioalkyl, heterocycloalkyl,heteroaryl, and heteroarylalkyl groups. In some yet additionalembodiments of the methods, the product having beta-glucose linkages isa steviol glycoside. In some further embodiments of the methods, theglycosyl group acceptor is rebaudioside D, the glycosyl group donor isADP-glucose, and the product having beta-glucose linkages isrebaudioside M. In some further embodiments of the methods, the glycosylgroup acceptor is stevioside, the glycosyl group donor is ADP-glucose,and the product having beta-glucose linkages is rebaudioside A orrebaudioside I.

The present invention also provides methods for production of nucleosidediphosphoglucose, comprising the steps of: providing a nucleosidediphosphate-dependent synthase, a nucleoside diphosphate, and adisaccharide, trisaccharide, or oligosaccharide substrate of thesynthase; contacting the synthase, nucleoside diphosphate, andsaccharide under conditions such that the saccharide is cleaved toproduce a lower molecular weight saccharide and nucleosidediphosphoglucose. In some embodiments of the method, this method iscombined with the previously described method. In some additionalembodiments of the method, the nucleoside diphosphate is ADP, thenucleoside diphosphoglucose is ADP-glucose, and the synthase substrateis sucrose.

DESCRIPTION OF THE DRAWINGS

FIG. 1 provides an enzymatic reaction scheme in which aglycosyltransferase catalyzes the transfer of a glucosyl group from anucleoside diphosphoglucose (NDP-glucose), for example ADP-glucose, toan acceptor, for example R—OH, where R is any glycosyl, alkoxy, carboxy,aminocarbonyl, heteroalkyl, heteroalkenyl, heteroalkynyl, carboxyalkyl,aminoalkyl, haloalkyl, alkylthioalkyl, heterocycloalkyl, heteroaryl, orheteroarylalkyl group. In a further embodiment, R—OH is a stevioside orrebaudioside D, and the product is rebaudioside A, rebaudioside I, orrebaudioside M. A nucleoside diphosphate dependent synthase catalyzesthe transfer of a glucosyl group from a glucose donor (e.g., sucrose),to a nucleoside diphosphate, regenerating NDP-glucose and releasing abyproduct (e.g., fructose).

FIG. 2 provides the structure of rebaudioside M with the carbonsnumbered.

FIG. 3 provides the structure of rebaudioside I with the carbonsnumbered.

DESCRIPTION OF THE INVENTION

The present invention provides engineered glycosyltransferase (GT)enzymes, polypeptides having GT activity, and polynucleotides encodingthese enzymes, as well as vectors and host cells comprising thesepolynucleotides and polypeptides. The present invention providesengineered sucrose synthase (SuS) enzymes, polypeptides having SuSactivity, and polynucleotides encoding these enzymes, as well as vectorsand host cells comprising these polynucleotides and polypeptides.

The present invention also provides compositions comprising the GTenzymes and methods of using the engineered GT enzymes to make productswith β-glucose linkages. The present invention further providescompositions and methods for the production of rebaudiosides (e.g.,rebaudioside M, rebaudioside A, rebaudioside I, and rebaudioside D). Thepresent invention also provides compositions comprising the SuS enzymesand methods of using them. Methods for producing GT and SuS enzymes arealso provided.

Unless defined otherwise, all technical and scientific terms used hereingenerally have the same meaning as commonly understood by one ofordinary skill in the art to which this invention pertains. Generally,the nomenclature used herein and the laboratory procedures of cellculture, molecular genetics, microbiology, organic chemistry, analyticalchemistry and nucleic acid chemistry described below are thosewell-known and commonly employed in the art. Such techniques arewell-known and described in numerous texts and reference works wellknown to those of skill in the art. Standard techniques, ormodifications thereof, are used for chemical syntheses and chemicalanalyses. All patents, patent applications, articles and publicationsmentioned herein, both supra and infra, are hereby expresslyincorporated herein by reference.

Although any suitable methods and materials similar or equivalent tothose described herein find use in the practice of the presentinvention, some methods and materials are described herein. It is to beunderstood that this invention is not limited to the particularmethodology, protocols, and reagents described, as these may vary,depending upon the context they are used by those of skill in the art.Accordingly, the terms defined immediately below are more fullydescribed by reference to the invention as a whole.

It is to be understood that both the foregoing general description andthe following detailed description are exemplary and explanatory onlyand are not restrictive of the present invention. The section headingsused herein are for organizational purposes only and not to be construedas limiting the subject matter described. Numeric ranges are inclusiveof the numbers defining the range. Thus, every numerical range disclosedherein is intended to encompass every narrower numerical range thatfalls within such broader numerical range, as if such narrower numericalranges were all expressly written herein. It is also intended that everymaximum (or minimum) numerical limitation disclosed herein includesevery lower (or higher) numerical limitation, as if such lower (orhigher) numerical limitations were expressly written herein.

Abbreviations

The abbreviations used for the genetically encoded amino acids areconventional and are as follows:

Three-Letter One-Letter Amino Acid Abbreviation Abbreviation Alanine AlaA Arginine Arg R Asparagine Asn N Aspartate Asp D Cysteine Cys CGlutamate Glu E Glutamine Gln Q Glycine Gly G Histidine His H IsoleucineIle I Leucine Leu L Lysine Lys K Methionine Met M Phenylalanine Phe FProline Pro P Serine Ser S Threonine Thr T Tryptophan Trp W Tyrosine TyrY Valine Val V

When the three-letter abbreviations are used, unless specificallypreceded by an “L” or a “D” or clear from the context in which theabbreviation is used, the amino acid may be in either the L- orD-configuration about α-carbon (C_(α)). For example, whereas “Ala”designates alanine without specifying the configuration about theα-carbon, “D-Ala” and “L-Ala” designate D-alanine and L-alanine,respectively. When the one-letter abbreviations are used, upper caseletters designate amino acids in the L-configuration about the α-carbonand lower case letters designate amino acids in the D-configurationabout the α-carbon. For example, “A” designates L-alanine and “a”designates D-alanine. When polypeptide sequences are presented as astring of one-letter or three-letter abbreviations (or mixturesthereof), the sequences are presented in the amino (N) to carboxy (C)direction in accordance with common convention.

The abbreviations used for the genetically encoding nucleosides areconventional and are as follows: adenosine (A); guanosine (G); cytidine(C); thymidine (T); and uridine (U). Unless specifically delineated, theabbreviated nucleosides may be either ribonucleosides or2′-deoxyribonucleosides. The nucleosides may be specified as beingeither ribonucleosides or 2′-deoxyribonucleosides on an individual basisor on an aggregate basis. When nucleic acid sequences are presented as astring of one-letter abbreviations, the sequences are presented in the5′ to 3′ direction in accordance with common convention, and thephosphates are not indicated.

Definitions

In reference to the present invention, the technical and scientificterms used in the descriptions herein will have the meanings commonlyunderstood by one of ordinary skill in the art, unless specificallydefined otherwise. Accordingly, the following terms are intended to havethe following meanings.

As used herein, the singular forms “a”, “an” and “the” include pluralreferents unless the context clearly indicates otherwise. Thus, forexample, reference to “a polypeptide” includes more than onepolypeptide.

Similarly, “comprise,” “comprises,” “comprising” “include,” “includes,”and “including” are interchangeable and not intended to be limiting.Thus, as used herein, the term “comprising” and its cognates are used intheir inclusive sense (i.e., equivalent to the term “including” and itscorresponding cognates).

It is to be further understood that where descriptions of variousembodiments use the term “comprising,” those skilled in the art wouldunderstand that in some specific instances, an embodiment can bealternatively described using language “consisting essentially of” or“consisting of.”

As used herein, the term “about” means an acceptable error for aparticular value. In some instances “about” means within 0.05%, 0.5%,1.0%, or 2.0%, of a given value range. In some instances, “about” meanswithin 1, 2, 3, or 4 standard deviations of a given value.

As used herein, “EC” number refers to the Enzyme Nomenclature of theNomenclature Committee of the International Union of Biochemistry andMolecular Biology (NC-IUBMB). The IUBMB biochemical classification is anumerical classification system for enzymes based on the chemicalreactions they catalyze.

As used herein, “ATCC” refers to the American Type Culture Collectionwhose biorepository collection includes genes and strains.

As used herein, “NCBI” refers to National Center for BiologicalInformation and the sequence databases provided therein.

“Protein,” “polypeptide,” and “peptide” are used interchangeably hereinto denote a polymer of at least two amino acids covalently linked by anamide bond, regardless of length or post-translational modification(e.g., glycosylation or phosphorylation). Included within thisdefinition are D- and L-amino acids, and mixtures of D- and L-aminoacids, as well as polymers comprising D- and L-amino acids, and mixturesof D- and L-amino acids.

“Amino acids” are referred to herein by either their commonly knownthree-letter symbols or by the one-letter symbols recommended byIUPAC-IUB Biochemical Nomenclature Commission.

Nucleotides, likewise, may be referred to by their commonly acceptedsingle letter codes.

As used herein, “polynucleotide” and “nucleic acid” refer to two or morenucleotides that are covalently linked together. The polynucleotide maybe wholly comprised of ribonucleotides (i.e., RNA), wholly comprised of2′ deoxyribonucleotides (i.e., DNA), or comprised of mixtures of ribo-and 2′ deoxyribonucleotides. While the nucleosides will typically belinked together via standard phosphodiester linkages, thepolynucleotides may include one or more non-standard linkages. Thepolynucleotide may be single-stranded or double-stranded, or may includeboth single-stranded regions and double-stranded regions. Moreover,while a polynucleotide will typically be composed of the naturallyoccurring encoding nucleobases (i.e., adenine, guanine, uracil, thymineand cytosine), it may include one or more modified and/or syntheticnucleobases, such as, for example, inosine, xanthine, hypoxanthine, etc.In some embodiments, such modified or synthetic nucleobases arenucleobases encoding amino acid sequences.

As used herein, “nucleoside” refers to glycosylamines comprising anucleobase (i.e., a nitrogenous base), and a 5-carbon sugar (e.g.,ribose or deoxyribose). Non-limiting examples of nucleosides includecytidine, uridine, adenosine, guanosine, thymidine, and inosine. Incontrast, the term “nucleotide” refers to the glycosylamines comprisinga nucleobase, a 5-carbon sugar, and one or more phosphate groups. Insome embodiments, nucleosides can be phosphorylated by kinases toproduce nucleotides.

As used herein, “nucleoside diphosphate” refers to glycosylaminescomprising a nucleobase (i.e., a nitrogenous base), a 5-carbon sugar(e.g., ribose or deoxyribose), and a diphosphate (i.e., pyrophosphate)moiety. In some embodiments herein, “nucleoside diphosphate” isabbreviated as “NDP.” Non-limiting examples of nucleoside diphosphatesinclude cytidine diphosphate (CDP), uridine diphosphate (UDP), adenosinediphosphate (ADP), guanosine diphosphate (GDP), thymidine diphosphate(TDP), and inosine diphosphate. The terms “nucleoside” and “nucleotide”may be used interchangeably in some contexts.

As used herein, “coding sequence” refers to that portion of a nucleicacid (e.g., a gene) that encodes an amino acid sequence of a protein.

As used herein, the terms “biocatalysis,” “biocatalytic,”“biotransformation,” and “biosynthesis” refer to the use of enzymes toperform chemical reactions on organic compounds.

As used herein, “glycosyltransferase” (GT) refers to a polypeptidehaving an enzymatic capability of transferring glycosyl residues from anactivated sugar donor to monomeric and polymeric acceptor molecules. Insome embodiments, the glycosyltransferases are referred to as“glycosyltransferase variants” or “glycosyltransferase combinatorialvariants.” In some embodiments, “glycosyltransferase” refers to anUDP-glucuronosyltransferase enzyme of the classification EC 2.4.1.17,which catalyzes the transfer of glucose from UDP-α-D-glucuronate (alsoknown as UDP-glucose) to an acceptor, releasing UDP and forming acceptorβ-D-glucuronoside. The Carbohydrate-Active Enzymes database (CAZy)provides a continuously updated list of the glycosyltransferasefamilies. In some embodiments, the glycosyltransferases include, but arenot limited to, enzymes classified in the GT1 family. In some preferredembodiments, the glycosyltransferase variants of the present inventionpreferentially utilize ADP-glucose. In some additional embodiments, theglycosyltransferase enzymes of the present invention do not utilizeUDP-glucose. In some further embodiments, the glycosyltransferasevariants of the present invention utilize ADP-glucose, CDP-glucose,TDP-glucose, GDP-glucose, and/or IDT-glucose, but not UDP-glucose. Thus,in some preferred embodiments, the present invention providesADP-glucose-dependent glycosyltransferases (ADP-glycosyltransferases;AGTs), CDP-glucose-dependent glycosyltransferases(CDP-glycosyltransferases; CGTs), GDP-glucose-dependentglycosyltransferases (GDP-glycosyltransferases; GGTs),TDP-glucose-dependent glycosyltransferases (TDP-glycosyltransferases;TGTs), and IDP-glucose-dependent glycosyltransferase(IDP-glycosyltransferases; IGTs).

As used herein, “NDP-glycosyltransferase” (NDP-GT) refers to apolypeptide having an enzymatic capability of transferring glycosylresidues from an activated sugar donor that is an NDP to monomeric andpolymeric acceptor molecules. In some embodiments,NDP-glycosyltransferases are generally referred to as“glycosyltransferases.” Indeed, the term “glycosyltransferase” as usedherein encompasses NDP-glycosyltransferases, including, but not limitedto ADP-glucose-dependent glycosyltransferases (ADP-glycosyltransferases;AGTs), CDP-glucose-dependent glycosyltransferases(CDP-glycosyltransferases; CGTs), GDP-glucose-dependentglycosyltransferase (GDP-glycosyltransferases; GGTs),TDP-glucose-dependent glycosyltransferases (TDP-glycosyltransferases;TGTs), and IDP-glucose-dependent glycosyltransferase(IDP-glycosyltransferases; IGTs). In some embodiments, theglycosyltransferase enzymes of the present invention utilizeADP-glucose, CDP-glucose, TDP-glucose, GDP-glucose, and/or IDT-glucose,but not UDP-glucose. In some additional embodiments the enzymes arereferred to as “variants” or “combinatorial variants” (e.g.,ADP-glycosyltransferase variants).

As used herein, “transglycosylation” refers to a reaction in which aglycosyl residue is transferred from a disaccharide, trisaccharide, oroligosaccharide donor to an aglycosylated or glycosylated acceptormolecule.

As used herein, “transglucosylation” refers to a transglycosylationreaction in which the glycosyl residue that is transferred is a glucoseand the disaccharide, trisaccharide, or oligosaccharide donor containsglucose.

As used herein, “glycosylation” refers to the formation of a glycosidiclinkage between a glycosyl residue and an acceptor molecule.

As used herein, “glucosylation” refers to the formation of a glycosidiclinkage between a glucose residue and an acceptor molecule.

As used herein, “glycosyl” refers to an organic group that is aunivalent free radical or substituent structure obtained by removing thehemiacetal hydroxyl group from the cyclic form of a monosaccharide,lower oligosaccharide or oligosaccharide derivative. Glycosyl groupsreact with inorganic acids (e.g., phosphoric acid) to form esters (e.g.,glucose 1-phosphate).

As used herein, “glycoside” refers to a molecule in which a carbohydrate(e.g., sugar) is bound to another functional group by a glycosidic bond.Glycosides can be hydrolyzed to produce a sugar and a non-sugar (i.e.,aglycone) component.

As used herein, the term “steviol glycoside” refers to a glycoside ofsteviol, including but not limited to, naturally occurring steviolglycosides (e.g., stevioside, steviolmonoside, steviolbioside,rubusoside, dulcoside B, dulcoside A, rebaudioside B, rebaudioside G,rebaudioside C, rebaudioside F, rebaudioside A, rebaudioside I,rebaudioside E, rebaudioside H, rebaudioside L, rebaudioside K,rebaudioside J, rebaudioside M (also referred to as rebaudioside X),rebaudioside D, rebaudioside N, rebaudioside O), and synthetic steviolglycosides (e.g., enzymatically glucosylated steviol glycosides), andcombinations thereof. The chemical structures of steviol and itsglycosides are below (See, WO 2013/176738).

As used herein, “stevioside substrate” refers to any suitable materialcomprising at least one steviol glycoside.

Chemical Structure of Steviol and Its Glycosides

Glycoside R₁ R₂ Steviol H H Steviolmonoside H Glcβ1- Steviol Glcβ1- Hmonoglucosyl ester Rubusoside Glcβ1- Glcβ1- Steviolbioside H Glcβ (1-2)Glcβ1- Dulcoside A Glcβ1- Rhaα(1-2) Glcβ1- Stevioside Glcβ1- Glcβ (1-2)Glcβ1- Rebaudioside B H Glcβ (1-2)[Glcβ (1-3)] Glcβ1- Rebaudioside CGlcβ1- Rhaα(1-2)[Glcβ (1-3)] Glcβ1- Rebaudioside A Glcβ1- Glcβ(1-2)[Glcβ (1-3)] Glcβ1- Rebaudioside D Glcβ (1-2) Glcβ1- Glcβ(1-2)[Glcβ (1-3)] Glcβ1- Rebaudioside I Glcβ (1-3) Glcβ1- Glcβ(1-2)[Glcβ (1-3)] Glcβ1- Rebaudioside M Glcβ (1-2)[Glcβ Glcβ (1-2)[Glcβ(1-3)] Glcβ1- (1-3)]Glcβ1- (Glc = glucose, Rha = rhamnose)

As used herein, “sucrose synthase” refers to aglycosyltransferase enzyme(EC 2.4.1.1.13) that reversibly catalyzes the chemical reactionNDP-glucose+D-fructose to NDP and sucrose. In some embodiments, thepresent invention provides variants of Acidithiobacillus caldus sucrosesynthase (“AcSuS”). In some embodiments, these enzymes are referred toas“sucrose synthase variants,” “SuS,” “SUS,” “SUS variants,” “SUSvariants,” “sucrose synthase combinatorial variants,” or “SuScombinatorial variants,” or “SUS combinatorial variants.” In someembodiments, these variants preferentially utilize NDPs other thanuridine (i.e., ADP-glucose, CDP-glucose, TDP-glucose, GDP-glucose,and/or IDP-glucose are utilized, rather than UDP-glucose). In someembodiments, these variants do not utilize UDP-glucose.

As used herein, the term “one-pot reaction” refers to the production ofrebaudioside of interest in one reaction vessel. In some embodiments,the term is used in reference to the production of rebM from a startingmaterial, including but not limited to asrebA and/or steviosides withthe intermediate production of other rebaudiosides (e.g., rebD and/orrebI). In some embodiments, the conversion of stevioside to RebA, RebAto RebD and/or RebI and RebD and/or RebI to Reb M, are conducted as amultiple enzyme cascade in one reaction vessel.

As used herein, “wild-type” and “naturally-occurring” refer to the formfound in nature. For example a wild-type polypeptide or polynucleotidesequence is a sequence present in an organism that can be isolated froma source in nature and which has not been intentionally modified byhuman manipulation.

As used herein, “recombinant,” “engineered,” and “non-naturallyoccurring” when used with reference to a cell, nucleic acid, orpolypeptide, refers to a material, or a material corresponding to thenatural or native form of the material, that has been modified in amanner that would not otherwise exist in nature. In some embodiments,the cell, nucleic acid or polypeptide is identical a naturally occurringcell, nucleic acid or polypeptide, but is produced or derived fromsynthetic materials and/or by manipulation using recombinant techniques.Non-limiting examples include, among others, recombinant cellsexpressing genes that are not found within the native (non-recombinant)form of the cell or express native genes that are otherwise expressed ata different level.

The term “percent (%) sequence identity” is used herein to refer tocomparisons among polynucleotides or polypeptides, and are determined bycomparing two optimally aligned sequences over a comparison window,wherein the portion of the polynucleotide or polypeptide sequence in thecomparison window may comprise additions or deletions (i.e., gaps) ascompared to the reference sequence for optimal alignment of the twosequences. The percentage may be calculated by determining the number ofpositions at which the identical nucleic acid base or amino acid residueoccurs in both sequences to yield the number of matched positions,dividing the number of matched positions by the total number ofpositions in the window of comparison and multiplying the result by 100to yield the percentage of sequence identity. Alternatively, thepercentage may be calculated by determining the number of positions atwhich either the identical nucleic acid base or amino acid residueoccurs in both sequences or a nucleic acid base or amino acid residue isaligned with a gap to yield the number of matched positions, dividingthe number of matched positions by the total number of positions in thewindow of comparison and multiplying the result by 100 to yield thepercentage of sequence identity. Those of skill in the art appreciatethat there are many established algorithms available to align twosequences. Optimal alignment of sequences for comparison can beconducted by any suitable method, including, but not limited to thelocal homology algorithm of Smith and Waterman (Smith and Waterman, Adv.Appl. Math., 2:482 [1981]), by the homology alignment algorithm ofNeedleman and Wunsch (Needleman and Wunsch, J. Mol. Biol., 48:443[1970]), by the search for similarity method of Pearson and Lipman(Pearson and Lipman, Proc. Natl. Acad. Sci. USA 85:2444 [1988]), bycomputerized implementations of these algorithms (e.g., GAP, BESTFIT,FASTA, and TFASTA in the GCG Wisconsin Software Package), or by visualinspection, as known in the art. Examples of algorithms that aresuitable for determining percent sequence identity and sequencesimilarity include, but are not limited to the BLAST and BLAST 2.0algorithms, which are described by Altschul et al. (See Altschul et al.,J. Mol. Biol., 215: 403-410 [1990]; and Altschul et al., Nucl. AcidsRes., 3389-3402 [1977], respectively). Software for performing BLASTanalyses is publicly available through the National Center forBiotechnology Information website. This algorithm involves firstidentifying high scoring sequence pairs (HSPs) by identifying shortwords of length W in the query sequence, which either match or satisfysome positive-valued threshold score T when aligned with a word of thesame length in a database sequence. T is referred to as, theneighborhood word score threshold (See, Altschul et al., supra). Theseinitial neighborhood word hits act as seeds for initiating searches tofind longer HSPs containing them. The word hits are then extended inboth directions along each sequence for as far as the cumulativealignment score can be increased. Cumulative scores are calculatedusing, for nucleotide sequences, the parameters M (reward score for apair of matching residues; always >0) and N (penalty score formismatching residues; always <0). For amino acid sequences, a scoringmatrix is used to calculate the cumulative score. Extension of the wordhits in each direction are halted when: the cumulative alignment scorefalls off by the quantity X from its maximum achieved value; thecumulative score goes to zero or below, due to the accumulation of oneor more negative-scoring residue alignments; or the end of eithersequence is reached. The BLAST algorithm parameters W, T, and Xdetermine the sensitivity and speed of the alignment. The BLASTN program(for nucleotide sequences) uses as defaults a wordlength (W) of 11, anexpectation (E) of 10, M=5, N=−4, and a comparison of both strands. Foramino acid sequences, the BLASTP program uses as defaults a wordlength(W) of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix(See, Henikoff and Henikoff, Proc. Natl. Acad. Sci. USA 89:10915[1989]). Exemplary determination of sequence alignment and % sequenceidentity can employ the BESTFIT or GAP programs in the GCG WisconsinSoftware package (Accelrys, Madison Wis.), using default parametersprovided.

As used herein, “reference sequence” refers to a defined sequence usedas a basis for a sequence and/or activity comparison. A referencesequence may be a subset of a larger sequence, for example, a segment ofa full-length gene or polypeptide sequence. Generally, a referencesequence is at least 20 nucleotide or amino acid residues in length, atleast 25 residues in length, at least 50 residues in length, at least100 residues in length or the full length of the nucleic acid orpolypeptide. Since two polynucleotides or polypeptides may each (1)comprise a sequence (i.e., a portion of the complete sequence) that issimilar between the two sequences, and (2) may further comprise asequence that is divergent between the two sequences, sequencecomparisons between two (or more) polynucleotides or polypeptides aretypically performed by comparing sequences of the two polynucleotides orpolypeptides over a “comparison window” to identify and compare localregions of sequence similarity. In some embodiments, a “referencesequence” can be based on a primary amino acid sequence, where thereference sequence is a sequence that can have one or more changes inthe primary sequence.

As used herein, “comparison window” refers to a conceptual segment of atleast about 20 contiguous nucleotide positions or amino acid residueswherein a sequence may be compared to a reference sequence of at least20 contiguous nucleotides or amino acids and wherein the portion of thesequence in the comparison window may comprise additions or deletions(i.e., gaps) of 20 percent or less as compared to the reference sequence(which does not comprise additions or deletions) for optimal alignmentof the two sequences. The comparison window can be longer than 20contiguous residues, and includes, optionally 30, 40, 50, 100, or longerwindows.

As used herein, “corresponding to,” “reference to,” and “relative to”when used in the context of the numbering of a given amino acid orpolynucleotide sequence refer to the numbering of the residues of aspecified reference sequence when the given amino acid or polynucleotidesequence is compared to the reference sequence. In other words, theresidue number or residue position of a given polymer is designated withrespect to the reference sequence rather than by the actual numericalposition of the residue within the given amino acid or polynucleotidesequence. For example, a given amino acid sequence, such as that of anengineered glycosyltransferase, can be aligned to a reference sequenceby introducing gaps to optimize residue matches between the twosequences. In these cases, although the gaps are present, the numberingof the residue in the given amino acid or polynucleotide sequence ismade with respect to the reference sequence to which it has beenaligned.

As used herein, “substantial identity” refers to a polynucleotide orpolypeptide sequence that has at least 80 percent sequence identity, atleast 85 percent identity, at least between 89 to 95 percent sequenceidentity, or more usually, at least 99 percent sequence identity ascompared to a reference sequence over a comparison window of at least 20residue positions, frequently over a window of at least 30-50 residues,wherein the percentage of sequence identity is calculated by comparingthe reference sequence to a sequence that includes deletions oradditions which total 20 percent or less of the reference sequence overthe window of comparison. In some specific embodiments applied topolypeptides, the term “substantial identity” means that two polypeptidesequences, when optimally aligned, such as by the programs GAP orBESTFIT using default gap weights, share at least 80 percent sequenceidentity, preferably at least 89 percent sequence identity, at least 95percent sequence identity or more (e.g., 99 percent sequence identity).In some embodiments, residue positions that are not identical insequences being compared differ by conservative amino acidsubstitutions.

As used herein, “amino acid difference” and “residue difference” referto a difference in the amino acid residue at a position of a polypeptidesequence relative to the amino acid residue at a corresponding positionin a reference sequence. In some cases, the reference sequence has ahistidine tag, but the numbering is maintained relative to theequivalent reference sequence without the histidine tag. The positionsof amino acid differences generally are referred to herein as “Xn,”where n refers to the corresponding position in the reference sequenceupon which the residue difference is based. For example, a “residuedifference at position X93 as compared to SEQ ID NO:4” refers to adifference of the amino acid residue at the polypeptide positioncorresponding to position 93 of SEQ ID NO:4. Thus, if the referencepolypeptide of SEQ ID NO:4 has a serine at position 93, then a “residuedifference at position X93 as compared to SEQ ID NO:4” an amino acidsubstitution of any residue other than serine at the position of thepolypeptide corresponding to position 93 of SEQ ID NO:4. In mostinstances herein, the specific amino acid residue difference at aposition is indicated as “XnY” where “Xn” specified the correspondingposition as described above, and “Y” is the single letter identifier ofthe amino acid found in the engineered polypeptide (i.e., the differentresidue than in the reference polypeptide). In some instances (e.g., inthe Tables presented in the Examples), the present invention alsoprovides specific amino acid differences denoted by the conventionalnotation “AnB”, where A is the single letter identifier of the residuein the reference sequence, “n” is the number of the residue position inthe reference sequence, and B is the single letter identifier of theresidue substitution in the sequence of the engineered polypeptide. Insome instances, a polypeptide of the present invention can include oneor more amino acid residue differences relative to a reference sequence,which is indicated by a list of the specified positions where residuedifferences are present relative to the reference sequence. In someembodiments, where more than one amino acid can be used in a specificresidue position of a polypeptide, the various amino acid residues thatcan be used are separated by a “/” (e.g., X307H/X307P or X307H/P). Theslash may also be used to indicate multiple substitutions within a givenvariant (i.e., there is more than one substitution present in a givensequence, such as in a combinatorial variant). In some embodiments, thepresent invention includes engineered polypeptide sequences comprisingone or more amino acid differences comprising conservative ornon-conservative amino acid substitutions. In some additionalembodiments, the present invention provides engineered polypeptidesequences comprising both conservative and non-conservative amino acidsubstitutions.

As used herein, “conservative amino acid substitution” refers to asubstitution of a residue with a different residue having a similar sidechain, and thus typically involves substitution of the amino acid in thepolypeptide with amino acids within the same or similar defined class ofamino acids. By way of example and not limitation, in some embodiments,an amino acid with an aliphatic side chain is substituted with anotheraliphatic amino acid (e.g., alanine, valine, leucine, and isoleucine);an amino acid with an hydroxyl side chain is substituted with anotheramino acid with an hydroxyl side chain (e.g., serine and threonine); anamino acids having aromatic side chains is substituted with anotheramino acid having an aromatic side chain (e.g., phenylalanine, tyrosine,tryptophan, and histidine); an amino acid with a basic side chain issubstituted with another amino acid with a basis side chain (e.g.,lysine and arginine); an amino acid with an acidic side chain issubstituted with another amino acid with an acidic side chain (e.g.,aspartic acid or glutamic acid); and/or a hydrophobic or hydrophilicamino acid is replaced with another hydrophobic or hydrophilic aminoacid, respectively.

As used herein, “non-conservative substitution” refers to substitutionof an amino acid in the polypeptide with an amino acid withsignificantly differing side chain properties. Non-conservativesubstitutions may use amino acids between, rather than within, thedefined groups and affects (a) the structure of the peptide backbone inthe area of the substitution (e.g., proline for glycine) (b) the chargeor hydrophobicity, or (c) the bulk of the side chain. By way of exampleand not limitation, an exemplary non-conservative substitution can be anacidic amino acid substituted with a basic or aliphatic amino acid; anaromatic amino acid substituted with a small amino acid; and ahydrophilic amino acid substituted with a hydrophobic amino acid.

As used herein, “deletion” refers to modification to the polypeptide byremoval of one or more amino acids from the reference polypeptide.Deletions can comprise removal of 1 or more amino acids, 2 or more aminoacids, 5 or more amino acids, 10 or more amino acids, 15 or more aminoacids, or 20 or more amino acids, up to 10% of the total number of aminoacids, or up to 20% of the total number of amino acids making up thereference enzyme while retaining enzymatic activity and/or retaining theimproved properties of an engineered glycosyltransferase enzyme.Deletions can be directed to the internal portions and/or terminalportions of the polypeptide. In various embodiments, the deletion cancomprise a continuous segment or can be discontinuous. Deletions aretypically indicated by “−” in amino acid sequences.

As used herein, “insertion” refers to modification to the polypeptide byaddition of one or more amino acids from the reference polypeptide.Insertions can be in the internal portions of the polypeptide, or to thecarboxy or amino terminus. Insertions as used herein include fusionproteins as is known in the art. The insertion can be a contiguoussegment of amino acids or separated by one or more of the amino acids inthe naturally occurring polypeptide.

A “functional fragment” and “biologically active fragment” are usedinterchangeably herein to refer to a polypeptide that has anamino-terminal and/or carboxy-terminal deletion(s) and/or internaldeletions, but where the remaining amino acid sequence is identical tothe corresponding positions in the sequence to which it is beingcompared (e.g., a full-length engineered glycosyltransferase of thepresent invention) and that retains substantially all of the activity ofthe full-length polypeptide.

As used herein, “isolated polypeptide” refers to a polypeptide which issubstantially separated from other contaminants that naturally accompanyit (e.g., protein, lipids, and polynucleotides). The term embracespolypeptides which have been removed or purified from theirnaturally-occurring environment or expression system (e.g., within ahost cell or via in vitro synthesis). The recombinantglycosyltransferase polypeptides may be present within a cell, presentin the cellular medium, or prepared in various forms, such as lysates orisolated preparations. As such, in some embodiments, the recombinantglycosyltransferase polypeptides can be an isolated polypeptide.

As used herein, “substantially pure polypeptide” or “purified protein”refers to a composition in which the polypeptide species is thepredominant species present (i.e., on a molar or weight basis it is moreabundant than any other individual macromolecular species in thecomposition), and is generally a substantially purified composition whenthe object species comprises at least about 50 percent of themacromolecular species present by mole or % weight. However, in someembodiments, the composition comprising glycosyltransferase comprisesglycosyltransferase that is less than 50% pure (e.g., about 10%, about20%, about 30%, about 40%, or about 50%) Generally, a substantially pureglycosyltransferase composition comprises about 60% or more, about 70%or more, about 80% or more, about 90% or more, about 95% or more, andabout 98% or more of all macromolecular species by mole or % weightpresent in the composition. In some embodiments, the object species ispurified to essential homogeneity (i.e., contaminant species cannot bedetected in the composition by conventional detection methods) whereinthe composition consists essentially of a single macromolecular species.Solvent species, small molecules (<500 Daltons), and elemental ionspecies are not considered macromolecular species. In some embodiments,the isolated recombinant glycosyltransferase polypeptides aresubstantially pure polypeptide compositions.

As used herein, “improved enzyme property” refers to at least oneimproved property of an enzyme. In some embodiments, the presentinvention provides engineered glycosyltransferase polypeptides thatexhibit an improvement in any enzyme property as compared to a referenceglycosyltransferase polypeptide and/or a wild-type glycosyltransferasepolypeptide, and/or another engineered glycosyltransferase polypeptide.Thus, the level of “improvement” can be determined and compared betweenvarious glycosyltransferase polypeptides, including wild-type, as wellas engineered glycosyltransferases. Improved properties include, but arenot limited, to such properties as increased protein expression,increased thermoactivity, increased thermostability, increased pHactivity, increased stability, increased enzymatic activity, increasedsubstrate specificity or affinity, increased specific activity,increased resistance to substrate or end-product inhibition, increasedchemical stability, improved chemoselectivity, improved solventstability, increased tolerance to acidic pH, increased tolerance toproteolytic activity (i.e., reduced sensitivity to proteolysis), reducedaggregation, increased solubility, and altered temperature profile. Inadditional embodiments, the term is used in reference to the at leastone improved property of sucrose synthase enzymes. In some embodiments,the present invention provides engineered sucrose synthase polypeptidesthat exhibit an improvement in any enzyme property as compared to areference sucrose synthase polypeptide and/or a wild-type sucrosesynthase polypeptide, and/or another engineered sucrose synthasepolypeptide. Thus, the level of “improvement” can be determined andcompared between various sucrose synthase polypeptides, includingwild-type, as well as engineered sucrose synthases.

As used herein, “increased enzymatic activity” and “enhanced catalyticactivity” refer to an improved property of the engineered polypeptides,which can be represented by an increase in specific activity (e.g.,product produced/time/weight protein) or an increase in percentconversion of the substrate to the product (e.g., percent conversion ofstarting amount of substrate to product in a specified time period usinga specified amount of enzyme) as compared to the reference enzyme. Insome embodiments, the terms refer to an improved property of engineeredglycosyltransferase polypeptides provided herein, which can berepresented by an increase in specific activity (e.g., productproduced/time/weight protein) or an increase in percent conversion ofthe substrate to the product (e.g., percent conversion of startingamount of substrate to product in a specified time period using aspecified amount of glycosyltransferase) as compared to the referenceglycosyltransferase enzyme. In some embodiments, the terms are used inreference to improved sucrose synthase enzymes provided herein.Exemplary methods to determine enzyme activity of the engineeredglycosyltransferases and sucrose synthases of the present invention areprovided in the Examples. Any property relating to enzyme activity maybe affected, including the classical enzyme properties of K_(m), V_(max)or k_(cat), changes of which can lead to increased enzymatic activity.For example, improvements in enzyme activity can be from about 1.1 foldthe enzymatic activity of the corresponding wild-type enzyme, to as muchas 2-fold, 5-fold, 10-fold, 20-fold, 25-fold, 50-fold, 75-fold,100-fold, 150-fold, 200-fold or more enzymatic activity than thenaturally occurring glycosyltransferase or another engineeredglycosyltransferase from which the glycosyltransferase polypeptides werederived.

As used herein, “conversion” refers to the enzymatic conversion (orbiotransformation) of a substrate(s) to the corresponding product(s).“Percent conversion” refers to the percent of the substrate that isconverted to the product within a period of time under specifiedconditions. Thus, the “enzymatic activity” or “activity” of aglycosyltransferase polypeptide can be expressed as “percent conversion”of the substrate to the product in a specific period of time.

Enzymes with “generalist properties” (or “generalist enzymes”) refer toenzymes that exhibit improved activity for a wide range of substrates,as compared to a parental sequence. Generalist enzymes do notnecessarily demonstrate improved activity for every possible substrate.In some embodiments, the present invention provides glycosyltransferasevariants with generalist properties, in that they demonstrate similar orimproved activity relative to the parental gene for a wide range ofsterically and electronically diverse substrates. In addition, thegeneralist enzymes provided herein were engineered to be improved acrossa wide range of diverse molecules to increase the production ofmetabolites/products.

The term “stringent hybridization conditions” is used herein to refer toconditions under which nucleic acid hybrids are stable. As known tothose of skill in the art, the stability of hybrids is reflected in themelting temperature (T_(m)) of the hybrids. In general, the stability ofa hybrid is a function of ion strength, temperature, G/C content, andthe presence of chaotropic agents. The T_(m) values for polynucleotidescan be calculated using known methods for predicting meltingtemperatures (See e.g., Baldino et al., Meth. Enzymol., 168:761-777[1989]; Bolton et al., Proc. Natl. Acad. Sci. USA 48:1390 [1962];Bresslauer et al., Proc. Natl. Acad. Sci. USA 83:8893-8897 [1986];Freier et al., Proc. Natl. Acad. Sci. USA 83:9373-9377 [1986]; Kierzeket al., Biochem., 25:7840-7846 [1986]; Rychlik et al., Nucl. Acids Res.,18:6409-6412 [1990] (erratum, Nucl. Acids Res., 19:698 [1991]); Sambrooket al., supra); Suggs et al., 1981, in Developmental Biology UsingPurified Genes, Brown et al. [eds.], pp. 683-693, Academic Press,Cambridge, Mass. [1981]; and Wetmur, Crit. Rev. Biochem. Mol. Biol.26:227-259 [1991]). In some embodiments, the polynucleotide encodes thepolypeptide disclosed herein and hybridizes under defined conditions,such as moderately stringent or highly stringent conditions, to thecomplement of a sequence encoding an engineered glycosyltransferaseenzyme of the present invention.

As used herein, “hybridization stringency” relates to hybridizationconditions, such as washing conditions, in the hybridization of nucleicacids. Generally, hybridization reactions are performed under conditionsof lower stringency, followed by washes of varying but higherstringency. The term “moderately stringent hybridization” refers toconditions that permit target-DNA to bind a complementary nucleic acidthat has about 60% identity, preferably about 75% identity, about 85%identity to the target DNA, with greater than about 90% identity totarget-polynucleotide. Exemplary moderately stringent conditions areconditions equivalent to hybridization in 50% formamide, 5×Denhart'ssolution, 5×SSPE, 0.2% SDS at 42° C., followed by washing in 0.2×SSPE,0.2% SDS, at 42° C. “High stringency hybridization” refers generally toconditions that are about 10° C. or less from the thermal meltingtemperature T_(m) as determined under the solution condition for adefined polynucleotide sequence. In some embodiments, a high stringencycondition refers to conditions that permit hybridization of only thosenucleic acid sequences that form stable hybrids in 0.018M NaCl at 65° C.(i.e., if a hybrid is not stable in 0.018M NaCl at 65° C., it will notbe stable under high stringency conditions, as contemplated herein).High stringency conditions can be provided, for example, byhybridization in conditions equivalent to 50% formamide, 5×Denhart'ssolution, 5×SSPE, 0.2% SDS at 42° C., followed by washing in 0.1×SSPE,and 0.1% SDS at 65° C. Another high stringency condition is hybridizingin conditions equivalent to hybridizing in 5×SSC containing 0.1% (w/v)SDS at 65° C. and washing in 0.1×SSC containing 0.1% SDS at 65° C. Otherhigh stringency hybridization conditions, as well as moderatelystringent conditions, are described in the references cited above.

As used herein, “codon optimized” refers to changes in the codons of thepolynucleotide encoding a protein to those preferentially used in aparticular organism such that the encoded protein is efficientlyexpressed in the organism of interest. Although the genetic code isdegenerate in that most amino acids are represented by several codons,called “synonyms” or “synonymous” codons, it is well known that codonusage by particular organisms is nonrandom and biased towards particularcodon triplets. This codon usage bias may be higher in reference to agiven gene, genes of common function or ancestral origin, highlyexpressed proteins versus low copy number proteins, and the aggregateprotein coding regions of an organism's genome. In some embodiments, thepolynucleotides encoding the glycosyltransferase enzymes may be codonoptimized for optimal production in the host organism selected forexpression.

As used herein, “preferred,” “optimal,” and “high codon usage bias”codons when used alone or in combination refer(s) interchangeably tocodons that are used at higher frequency in the protein coding regionsthan other codons that code for the same amino acid. The preferredcodons may be determined in relation to codon usage in a single gene, aset of genes of common function or origin, highly expressed genes, thecodon frequency in the aggregate protein coding regions of the wholeorganism, codon frequency in the aggregate protein coding regions ofrelated organisms, or combinations thereof. Codons whose frequencyincreases with the level of gene expression are typically optimal codonsfor expression. A variety of methods are known for determining the codonfrequency (e.g., codon usage, relative synonymous codon usage) and codonpreference in specific organisms, including multivariate analysis, forexample, using cluster analysis or correspondence analysis, and theeffective number of codons used in a gene (See e.g., GCGCodonPreference, Genetics Computer Group Wisconsin Package; CodonW,Peden, University of Nottingham; McInerney, Bioinform., 14:372-73[1998]; Stenico et al., Nucl. Acids Res., 222437-46 [1994]; and Wright,Gene 87:23-29 [1990]). Codon usage tables are available for manydifferent organisms (See e.g., Wada et al., Nucl. Acids Res.,20:2111-2118 [1992]; Nakamura et al., Nucl. Acids Res., 28:292 [2000];Duret, et al., supra; Henaut and Danchin, in Escherichia coli andSalmonella, Neidhardt, et al. (eds.), ASM Press, Washington D.C., p.2047-2066 [1996]). The data source for obtaining codon usage may rely onany available nucleotide sequence capable of coding for a protein. Thesedata sets include nucleic acid sequences actually known to encodeexpressed proteins (e.g., complete protein coding sequences-CDS),expressed sequence tags (ESTS), or predicted coding regions of genomicsequences (See e.g., Mount, Bioinformatics: Sequence and GenomeAnalysis, Chapter 8, Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y. [2001]; Uberbacher, Meth. Enzymol., 266:259-281 [1996]; andTiwari et al., Comput. Appl. Biosci., 13:263-270 [1997]).

As used herein, “control sequence” includes all components, which arenecessary or advantageous for the expression of a polynucleotide and/orpolypeptide of the present invention. Each control sequence may benative or foreign to the nucleic acid sequence encoding the polypeptide.Such control sequences include, but are not limited to, a leader,polyadenylation sequence, propeptide sequence, promoter sequence, signalpeptide sequence, initiation sequence and transcription terminator. At aminimum, the control sequences include a promoter, and transcriptionaland translational stop signals. The control sequences may be providedwith linkers for the purpose of introducing specific restriction sitesfacilitating ligation of the control sequences with the coding region ofthe nucleic acid sequence encoding a polypeptide.

“Operably linked” is defined herein as a configuration in which acontrol sequence is appropriately placed (i.e., in a functionalrelationship) at a position relative to a polynucleotide of interestsuch that the control sequence directs or regulates the expression ofthe polynucleotide and/or polypeptide of interest.

“Promoter sequence” refers to a nucleic acid sequence that is recognizedby a host cell for expression of a polynucleotide of interest, such as acoding sequence. The promoter sequence contains transcriptional controlsequences, which mediate the expression of a polynucleotide of interest.The promoter may be any nucleic acid sequence which showstranscriptional activity in the host cell of choice including mutant,truncated, and hybrid promoters, and may be obtained from genes encodingextracellular or intracellular polypeptides either homologous orheterologous to the host cell.

The phrase “suitable reaction conditions” refers to those conditions inthe enzymatic conversion reaction solution (e.g., ranges of enzymeloading, substrate loading, temperature, pH, buffers, co-solvents, etc.)under which a glycosyltransferase polypeptide of the present inventionis capable of converting a substrate to the desired product compound.Some exemplary “suitable reaction conditions” are provided herein.

As used herein, “loading,” such as in “compound loading” or “enzymeloading” refers to the concentration or amount of a component in areaction mixture at the start of the reaction.

As used herein, “substrate” in the context of an enzymatic conversionreaction process refers to the compound or molecule acted on by theengineered enzymes provided herein (e.g., engineered glycosyltransferasepolypeptides).

As used herein, the terms “biomass,” “biomass substrate,” “cellulosicbiomass,” “cellulosic feedstock,” and “cellulosic substrate” refer toany materials that contain cellulose. Biomass can be derived fromplants, animals, or microorganisms, and may include, but is not limitedto agricultural, industrial, and forestry residues, industrial andmunicipal wastes, and terrestrial and aquatic crops grown for energypurposes. Examples of cellulosic substrates include, but are not limitedto, wood, wood pulp, paper pulp, corn fiber, corn grain, corn cobs, cropresidues such as corn husks, corn stover, grasses, wheat, wheat straw,barley, barley straw, hay, rice, rice straw, switchgrass, waste paper,paper and pulp processing waste, woody or herbaceous plants, fruit orvegetable pulp, corn cobs, distillers grain, grasses, rice hulls,cotton, hemp, flax, sisal, sugar cane bagasse, sorghum, soy,switchgrass, components obtained from milling of grains, trees,branches, roots, leaves, wood chips, sawdust, shrubs and bushes,vegetables, fruits, and flowers and any suitable mixtures thereof Insome embodiments, the cellulosic biomass comprises, but is not limitedto cultivated crops (e.g., grasses, including C4 grasses, such as switchgrass, cord grass, rye grass, miscanthus, reed canary grass, or anycombination thereof), sugar processing residues, for example, but notlimited to, bagasse (e.g., sugar cane bagasse, beet pulp [e.g., sugarbeet], or a combination thereof), agricultural residues (e.g., soybeanstover, corn stover, corn fiber, rice straw, sugar cane straw, rice,rice hulls, barley straw, corn cobs, wheat straw, canola straw, oatstraw, oat hulls, corn fiber, hemp, flax, sisal, cotton, or anycombination thereof), fruit pulp, vegetable pulp, distillers' grains,forestry biomass (e.g., wood, wood pulp, paper pulp, recycled wood pulpfiber, sawdust, hardwood, such as aspen wood, softwood, or a combinationthereof). Furthermore, in some embodiments, the cellulosic biomasscomprises cellulosic waste material and/or forestry waste materials,including but not limited to, paper and pulp processing waste,newsprint, cardboard and the like. In some embodiments, the cellulosicbiomass comprises one species of fiber, while in some alternativeembodiments, the cellulosic biomass comprises a mixture of fibers thatoriginate from different cellulosic biomasses. In some embodiments, thebiomass may also comprise transgenic plants that express ligninaseand/or cellulase enzymes (See e.g., US 2008/0104724, incorporated byreference herein).

As used herein, the term “slurry” refers to an aqueous solution in whichare dispersed one or more solid components, such as a cellulosicsubstrate.

As used herein, “increasing” yield of a product (e.g., a steviolglycoside) from a reaction occurs when a particular component presentduring the reaction (e.g., a GH enzyme) causes more product to beproduced, compared with a reaction conducted under the same conditionswith the same substrate and other substituents, but in the absence ofthe component of interest.

As used herein, “hydrolyzing” cellulose or other polysaccharide occurswhen at least some of the glycosidic bonds between two monosaccharidespresent in the substrate are hydrolyzed, thereby detaching from eachother the two monomers that were previously bonded.

A reaction is said to be “substantially free” of a particular enzyme ifthe amount of that enzyme compared with other enzymes that participatein catalyzing the reaction is less than about 2%, about 1%, or about0.1% (wt/wt).

As used herein, “fractionating” a liquid (e.g., a culture broth) meansapplying a separation process (e.g., salt precipitation, columnchromatography, size exclusion, and filtration) or a combination of suchprocesses to provide a solution in which a desired protein (e.g.,rebaudiosides) comprises a greater percentage of total protein in thesolution than in the initial liquid product.

As used herein, “starting composition” refers to any composition thatcomprises at least one substrate. In some embodiments, the startingcomposition comprises any cellulosic substrate.

In some alternative embodiments, the term “starting composition” refersto any composition comprising at least one steviol glycoside, whereinone or more of the steviol glycosides act as substrate(s) for abiotransformation. In some embodiments, the starting composition isprovided as an aqueous solution. In some embodiments, the startingcomposition comprises at least one steviol glycoside selected fromstevioside, steviolmonoside, steviolbioside, rubusoside, dulcoside B,dulcoside A, rebaudioside B, rebaudioside G, rebaudioside C,rebaudioside F, rebaudioside A, rebaudioside I, rebaudioside E,rebaudioside H, rebaudioside L, rebaudioside K, rebaudioside J,rebaudioside M (also referred to as rebaudioside X), rebaudioside D,rebaudioside N, rebaudioside O, and synthetic steviol glycosides (e.g.,enzymatically glucosylated steviol glycosides), In some embodiments, thestarting composition comprises two or more steviol glycosides. In someembodiments, the starting composition comprises an extract obtained frompurification of Stevia rebaudiana plant material (e.g., leaves). In somealternative embodiments, the starting composition comprises commerciallyavailable stevia extract(s). Additional starting compositions compriseby-products of processes used to isolate and purify steviol glycosides.In some embodiments, the starting composition comprises purified orpartially purified steviol glycoside substrate(s). In some embodiments,the starting composition comprises greater than about 99% of aparticular steviol glycoside by weight.

In some embodiments, the starting composition comprises at least oneglycoside and a cellulosic component as the substrate to produce atleast one steviol glycoside (e.g., rebaudioside A, D, etc.).

As used herein, “product” in the context of an enzymatic conversionprocess refers to the compound or molecule resulting from the action ofan enzymatic polypeptide on a substrate. As used herein, in someembodiments, the term refers to the compound or molecule resulting fromthe action of the glycosyltransferase polypeptide on a substrate. Insome embodiments, the product provided by the present invention is asteviol glycoside. In some embodiments, the product comprises at leastone steviol glycoside selected from stevioside, steviolmonoside,steviolbioside, rubusoside, dulcoside B, dulcoside A, rebaudioside B,rebaudioside G, rebaudioside C, rebaudioside F, rebaudioside A,rebaudioside I, rebaudioside E, rebaudioside H, rebaudioside L,rebaudioside K, rebaudioside J, rebaudioside M (also referred to asrebaudioside X), rebaudioside D, rebaudioside N, rebaudioside O, andsynthetic steviol glycosides (e.g., enzymatically glucosylated steviolglycosides),

As used herein the term “culturing” refers to the growing of apopulation of microbial cells under any suitable conditions (e.g., usinga liquid, gel or solid medium).

Recombinant polypeptides can be produced using any suitable methodsknown in the art. Genes encoding the wild-type polypeptide of interestcan be cloned in vectors, such as plasmids, and expressed in desiredhosts, such as E. coli, etc. Variants of recombinant polypeptides can begenerated by various methods known in the art. Indeed, there is a widevariety of different mutagenesis techniques well known to those skilledin the art. In addition, mutagenesis kits are also available from manycommercial molecular biology suppliers. Methods are available to makespecific substitutions at defined amino acids (site-directed), specificor random mutations in a localized region of the gene (regio-specific),or random mutagenesis over the entire gene (e.g., saturationmutagenesis). Numerous suitable methods are known to those in the art togenerate enzyme variants, including but not limited to site-directedmutagenesis of single-stranded DNA or double-stranded DNA using PCR,cassette mutagenesis, gene synthesis, error-prone PCR, shuffling, andchemical saturation mutagenesis, or any other suitable method known inthe art. Mutagenesis and directed evolution methods can be readilyapplied to enzyme-encoding polynucleotides to generate variant librariesthat can be expressed, screened, and assayed. Any suitable mutagenesisand directed evolution methods find use in the present invention and arewell known in the art (See e.g., U.S. Pat. Nos. 5,605,793, 5,811,238,5,830,721, 5,834,252, 5,837,458, 5,928,905, 6,096,548, 6,117,679,6,132,970, 6,165,793, 6,180,406, 6,251,674, 6,265,201, 6,277,638,6,287,861, 6,287,862, 6,291,242, 6,297,053, 6,303,344, 6,309,883,6,319,713, 6,319,714, 6,323,030, 6,326,204, 6,335,160, 6,335,198,6,344,356, 6,352,859, 6,355,484, 6,358,740, 6,358,742, 6,365,377,6,365,408, 6,368,861, 6,372,497, 6,337,186, 6,376,246, 6,379,964,6,387,702, 6,391,552, 6,391,640, 6,395,547, 6,406,855, 6,406,910,6,413,745, 6,413,774, 6,420,175, 6,423,542, 6,426,224, 6,436,675,6,444,468, 6,455,253, 6,479,652, 6,482,647, 6,483,011, 6,484,105,6,489,146, 6,500,617, 6,500,639, 6,506,602, 6,506,603, 6,518,065,6,519,065, 6,521,453, 6,528,311, 6,537,746, 6,573,098, 6,576,467,6,579,678, 6,586,182, 6,602,986, 6,605,430, 6,613,514, 6,653,072,6,686,515, 6,703,240, 6,716,631, 6,825,001, 6,902,922, 6,917,882,6,946,296, 6,961,664, 6,995,017, 7,024,312, 7,058,515, 7,105,297,7,148,054, 7,220,566, 7,288,375, 7,384,387, 7,421,347, 7,430,477,7,462,469, 7,534,564, 7,620,500, 7,620,502, 7,629,170, 7,702,464,7,747,391, 7,747,393, 7,751,986, 7,776,598, 7,783,428, 7,795,030,7,853,410, 7,868,138, 7,783,428, 7,873,477, 7,873,499, 7,904,249,7,957,912, 7,981,614, 8,014,961, 8,029,988, 8,048,674, 8,058,001,8,076,138, 8,108,150, 8,170,806, 8,224,580, 8,377,681, 8,383,346,8,457,903, 8,504,498, 8,589,085, 8,762,066, 8,768,871, 9,593,326, andall related US, as well as PCT and non-US counterparts; Ling et al.,Anal. Biochem., 254(2):157-78 [1997]; Dale et al., Meth. Mol. Biol.,57:369-74 [1996]; Smith, Ann. Rev. Genet., 19:423-462 [1985]; Botsteinet al., Science, 229:1193-1201 [1985]; Carter, Biochem. J., 237:1-7[1986]; Kramer et al., Cell, 38:879-887 [1984]; Wells et al., Gene,34:315-323 [1985]; Minshull et al., Curr. Op. Chem. Biol., 3:284-290[1999]; Christians et al., Nat. Biotechnol., 17:259-264 [1999]; Crameriet al., Nature, 391:288-291 [1998]; Crameri, et al., Nat. Biotechnol.,15:436-438 [1997]; Zhang et al., Proc. Nat. Acad. Sci. U.S.A.,94:4504-4509 [1997]; Crameri et al., Nat. Biotechnol., 14:315-319[1996]; Stemmer, Nature, 370:389-391 [1994]; Stemmer, Proc. Nat. Acad.Sci. USA, 91:10747-10751 [1994]; WO 95/22625; WO 97/0078; WO 97/35966;WO 98/27230; WO 00/42651; WO 01/75767; and WO 2009/152336, all of whichare incorporated herein by reference).

In some embodiments, the enzyme clones obtained following mutagenesistreatment are screened by subjecting the enzyme preparations to adefined temperature (or other assay conditions) and measuring the amountof enzyme activity remaining after heat treatments or other suitableassay conditions. Clones containing a polynucleotide encoding apolypeptide are then isolated from the gene, sequenced to identify thenucleotide sequence changes (if any), and used to express the enzyme ina host cell. Measuring enzyme activity from the expression libraries canbe performed using any suitable method known in the art (e.g., standardbiochemistry techniques, such as HPLC analysis).

After the variants are produced, they can be screened for any desiredproperty (e.g., high or increased activity, or low or reduced activity,increased thermal activity, increased thermal stability, and/or acidicpH stability, etc.). In some embodiments, “recombinantglycosyltransferase polypeptides” (also referred to herein as“engineered glycosyltransferase polypeptides,” “variantglycosyltransferase enzymes,” “glycosyltransferase variants,” and“glycosyltransferase combinatorial variants”) find use. In someembodiments, “recombinant sucrose synthase polypeptides” (also referredto as “engineered sucrose synthase polypeptides,” “variant sucrosesynthase enzymes,” “sucrose synthase variants,” and “sucrose synthasecombinatorial variants”) find use.

As used herein, a “vector” is a DNA construct for introducing a DNAsequence into a cell. In some embodiments, the vector is an expressionvector that is operably linked to a suitable control sequence capable ofeffecting the expression in a suitable host of the polypeptide encodedin the DNA sequence. In some embodiments, an “expression vector” has apromoter sequence operably linked to the DNA sequence (e.g., transgene)to drive expression in a host cell, and in some embodiments, alsocomprises a transcription terminator sequence.

As used herein, the term “expression” includes any step involved in theproduction of the polypeptide including, but not limited to,transcription, post-transcriptional modification, translation, andpost-translational modification. In some embodiments, the term alsoencompasses secretion of the polypeptide from a cell.

As used herein, the term “produces” refers to the production of proteinsand/or other compounds by cells. It is intended that the term encompassany step involved in the production of polypeptides including, but notlimited to, transcription, post-transcriptional modification,translation, and post-translational modification. In some embodiments,the term also encompasses secretion of the polypeptide from a cell.

As used herein, an amino acid or nucleotide sequence (e.g., a promotersequence, signal peptide, terminator sequence, etc.) is “heterologous”to another sequence with which it is operably linked if the twosequences are not associated in nature. For example a “heterologouspolynucleotide” is any polynucleotide that is introduced into a hostcell by laboratory techniques, and includes polynucleotides that areremoved from a host cell, subjected to laboratory manipulation, and thenreintroduced into a host cell.

As used herein, the terms “host cell” and “host strain” refer tosuitable hosts for expression vectors comprising DNA provided herein(e.g., the polynucleotides encoding the glycosyltransferase variants).In some embodiments, the host cells are prokaryotic or eukaryotic cellsthat have been transformed or transfected with vectors constructed usingrecombinant DNA techniques as known in the art.

The term “analogue” means a polypeptide having more than 70% sequenceidentity but less than 100% sequence identity (e.g., more than 75%, 78%,80%, 83%, 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%sequence identity) with a reference polypeptide. In some embodiments,analogues means polypeptides that contain one or more non-naturallyoccurring amino acid residues including, but not limited, tohomoarginine, ornithine and norvaline, as well as naturally occurringamino acids. In some embodiments, analogues also include one or moreD-amino acid residues and non-peptide linkages between two or more aminoacid residues.

The term “effective amount” means an amount sufficient to produce thedesired result. One of general skill in the art may determine what theeffective amount by using routine experimentation.

The terms “isolated” and “purified” are used to refer to a molecule(e.g., an isolated nucleic acid, polypeptide, etc.) or other componentthat is removed from at least one other component with which it isnaturally associated. The term “purified” does not require absolutepurity, rather it is intended as a relative definition.

As used herein, “stereoselectivity” refers to the preferential formationin a chemical or enzymatic reaction of one stereoisomer over another.Stereoselectivity can be partial, where the formation of onestereoisomer is favored over the other, or it may be complete where onlyone stereoisomer is formed. When the stereoisomers are enantiomers, thestereoselectivity is referred to as enantioselectivity, the fraction(typically reported as a percentage) of one enantiomer in the sum ofboth. It is commonly alternatively reported in the art (typically as apercentage) as the enantiomeric excess (“e.e.”) calculated therefromaccording to the formula [major enantiomer−minor enantiomer]/[majorenantiomer+minor enantiomer]. Where the stereoisomers arediastereoisomers, the stereoselectivity is referred to asdiastereoselectivity, the fraction (typically reported as a percentage)of one diastereomer in a mixture of two diastereomers, commonlyalternatively reported as the diastereomeric excess (“d.e.”).Enantiomeric excess and diastereomeric excess are types of stereomericexcess.

As used herein, “regioselectivity” and “regioselective reaction” referto a reaction in which one direction of bond making or breaking occurspreferentially over all other possible directions. Reactions cancompletely (100%) regioselective if the discrimination is complete,substantially regioselective (at least 75%), or partially regioselective(x %, wherein the percentage is set dependent upon the reaction ofinterest), if the product of reaction at one site predominates over theproduct of reaction at other sites.

As used herein, “thermostable” refers to a glycosyltransferasepolypeptide that maintains similar activity (more than 60% to 80% forexample) after exposure to elevated temperatures (e.g., 40-80° C.) for aperiod of time (e.g., 0.5-24 h) compared to the wild-type enzyme exposedto the same elevated temperature.

As used herein, “solvent stable” refers to a glycosyltransferasepolypeptide that maintains similar activity (more than e.g., 60% to 80%)after exposure to varying concentrations (e.g., 5-99%) of solvent(ethanol, isopropyl alcohol, dimethylsulfoxide [DMSO], tetrahydrofuran,2-methyltetrahydrofuran, acetone, toluene, butyl acetate, methyltert-butyl ether, etc.) for a period of time (e.g., 0.5-24 h) comparedto the wild-type enzyme exposed to the same concentration of the samesolvent.

As used herein, “thermo- and solvent stable” refers to aglycosyltransferase polypeptide that is both thermostable and solventstable.

As used herein, “reductant” refers to a compound or agent capable ofconverting Fe⁺³ to Fe⁺². An exemplary reductant is ascorbic acid, whichis generally in the form of L-ascorbic acid.

As used herein, “optional” and “optionally” mean that the subsequentlydescribed event or circumstance may or may not occur, and that thedescription includes instances where the event or circumstance occursand instances in which it does not. One of ordinary skill in the artwould understand that with respect to any molecule described ascontaining one or more optional substituents, only sterically practicaland/or synthetically feasible compounds are meant to be included.“Optionally substituted” refers to all subsequent modifiers in a term orseries of chemical groups.

Glycosylation

Glycosylation can alter many properties of natural and syntheticproducts including stability, pharmacodynamics, solubility, and membranetransport. The present invention provides compositions, methods andenzymes suitable for generating new glycosylated compounds from variousaglycone and glycosylated substrates. In some embodiments, the presentinvention provides means to efficiently generate known glycosylatedcompounds from easily obtained precursors. In some cases, glycosylationis achieved through chemical synthesis methods. However, these methodstypically require undesirable chemicals and processes and can result inmixed products (e.g., with linkages in incorrect positions and/or withundesired anomeric configurations). Furthermore, carbohydrate chemistryrequires multiple protection and deprotection steps.

In contrast, glycosylating enzymes can be active under mild conditionsand can confer high positional selectivity and stereospecificity in asingle step. Many naturally-occurring glycosylated metabolites aregenerated in vivo using glycosyltransferases that transfer sugarmoieties from various sugar nucleosides. Many molecules, including manysecondary metabolites with antimicrobial, antitumor, natural sweetnessproperties, etc., comprise non-ribosomal peptide, polyketide, orterpenoid backbones modified with β-glycosidic linkages. Many of thediterpene glycosides extracted from the plant, Stevia rebaudianaBertoni, contain β-linked glucose molecules. Naturally, these moleculesare glycosylated in vivo using UDP-glucose dependent glycosyltransferase enzymes. The present invention provides a method (See, FIG.1), in which a new engineered glycosyltransferase is used to transferthe glucose moiety from a nucleoside diphosphoglucose to a substrate(e.g., rebaudioside D or stevioside), to produce one or more β-glucoselinked products (e.g., rebaudioside M, rebaudioside A, or rebaudiosideI). However, when used in vitro, the UDP-glucose can be prohibitivelyexpensive and/or unavailable. In the some additional embodiments, asynthase (e.g., sucrose synthase or trehalose synthase) acts in thereverse direction to form a nucleoside diphosphoglucose compound from anucleoside diphosphate and a glucose donor (e.g., sucrose, trehalose, orstarch).

Thus, glycosylation finds use in the production of natural sweeteners,such as those derived from the sweet herb, Stevia rebaudiana Bertoni. Asindicated above, this plant produces a number of diterpene glycosideswhich feature high intensity sweetness and sensory properties superiorto those of many other high potency sweeteners. The above-mentionedsweet glycosides, have a common aglycone (i.e., steviol), and differ bythe number and type of carbohydrate residues at the C13 and C19positions. Steviol glycosides differ from each other not only in theirmolecular structure, but also by their taste properties. Usually,stevioside is reported to be 89-143 times sweeter than sucrose, whilerebaudioside A is reported to be between 85 and 242 times sweeter thansucrose (See e.g., Kasai et al., Nippon Kagaku Kaishi, 1981:726-735[1981]). Of these common compounds, rebaudioside A has the leastastringent, the least bitter, and the least persistent aftertaste. Thus,it has the most favorable sensory attributes of the major steviolglycosides and has been commercialized. However, rebaudioside A onlyconstitutes a smaller fraction (about 20%) of total glycosides isolatedfrom Stevia rebaudiana Bertoni, with stevioside (about 70%) and minorsteviol glycosides making up the rest (See e.g., FAO, Chemical andTechnical Assessment, 63^(rd) JECFA, Steviol Glycosides [2004]). Thenaturally occurring but even less abundant compound rebaudioside M, alsoknown as rebaudioside X, is 200-350 times sweeter than sucrose and has areduced aftertaste relative to rebaudioside A (See e.g., Prakash et al.,Food, 3:162-175 [2014]). Thus, there is interest in thecommercialization of rebaudioside M, for example as a natural sweetener,but currently no viable commercial route to synthesize this compound.

Engineered Glycosyltransferase Polypeptides

The present invention provides glycosyltransferase polypeptides,polynucleotides encoding the polypeptides, methods of preparing thepolypeptides, and methods for using the polypeptides. Where thedescription relates to polypeptides, it is to be understood that it alsodescribes the polynucleotides encoding the polypeptides. In someembodiments, the present invention provides engineered, non-naturallyoccurring GT enzymes with improved properties as compared to wild-typeGT enzymes. Any suitable reaction conditions find use in the presentinvention. In some embodiments, methods are used to analyze the improvedproperties of the engineered polypeptides to carry out the transferasereaction. In some embodiments, the reaction conditions are modified withregard to concentrations or amounts of polypeptide, substrate,co-substrate, buffer, co-solvent, pH, conditions including temperatureand reaction time, and/or conditions with the polypeptide immobilized ona solid support, as further described below and in the Examples.

In some embodiments, the engineered GT polypeptides described hereinhave improved properties as compared to wild-type GT enzymes such as inthe conversion of steviol glycosides to further glycosylated steviolglycosides (e.g., stevioside to rebaudioside A or rebaudioside D torebaudioside M) and in the use of adenine diphosphoglucose or othernucleoside diphosphates. In some embodiments, the engineered GT enzymescomprise amino acid sequences having one or more residue differences ascompared to SEQ ID NO: 4, 8, 32, 232, 348, 548, 562, 696, 758, 770, 792,954, 1002,1054, 2600, 2718, 2814, 2884, 3016, 3082, 3244, 3346, 3502,3696, 4256, 4684, 4838, 4876, 5066, 5290, 5372, 5562, 5708, 5976, 6138,6288, 6468, 684, 7388, and/or 8088. In some embodiments, the engineeredGT enzymes are beta-1,2 glycosyltransferase variants having one or moreresidue differences as compared to SEQ ID NO: 758, 770, 792, 954, 1002,1054, 2600, 2718, 2814, 2884, 3016, 3082, 3244, 3346, 3502, 3696, 3956,4256, 4550, 7324, and/or 7784. In some embodiments, the engineered GTenzymes are beta-1,3 glycosyltransferase variants having one or moreresidue differences as compared to SEQ ID NO: 4, 8, 32, 232, 348, 548,562, 696, 758, 770, 792, 954, 1002, 1054, 2600, 2718, 2814, 2884, 3016,3082, 3244, 3346, 3502, 3696, 3956, 4256, 4550, 4684, 4838, 4876, 5066,5290, 5372, 5562, 5708, 5976, 6138, 6288, 6468, 6864, 7324, 7388, and/or8088. In some further embodiments, engineered GT polypeptides arecircular permuted proteins in which a peptide linker is incorporated atthe genetic level between the N- and C-termini and new amino acidpositions are selected as the location of the new N- and C-termini. Insome embodiments, the circular permuted GT enzymes comprise amino acidsequences having one or more residue differences as compared to SEQ IDNO: 4, 8, 32, 232, 348, 548, 562, 696, 758, 770, 792, 954, 1002, 1054,2600, 2718, 2814, 2884, 3016, 3082, 3244, 3346, 3502, 3696, 3956, 4256,4550, 4684, 4838, 4876, 5066, 5290, 5372, 5562, 5708, 5976, 6138, 6288,6468, 6864, 7324, 7388, 7784, and/or 8088. In some embodiments, thecircular permuted GT enzymes comprise amino acid sequences having one ormore residue differences as compared to SEQ ID NO: 32. In someembodiments, the GT enzymes comprise at least one sequence set forthherein. In some embodiments, the present invention provides engineeredglycosyltransferases comprising polypeptide sequences that have at least90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequenceidentity to SEQ ID NOS: 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28,30, 22, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64,66, 68, 70, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102,104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130,132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158,160, 162, 164, 166, 168, 170, 172, 174, 176, 178, 180, 182, 184, 186,188, 190, 192, 194, 196, 198, 200, 202, 204, 206, 208, 210, 212, 214,216, 218, 220, 222, 224, 226, 228, 230, 232, 234, 236, 238, 240, 242,244, 246, 248, 250, 252, 254, 256, 258, 260, 262, 264, 266, 268, 270,272, 274, 276, 278, 280, 282, 284, 286, 288, 290, 292, 294, 296, 298,300, 302, 304, 306, 308, 310, 312, 314, 316, 318, 320, 322, 324, 326,328, 330, 332, 334, 336, 338, 340, 342, 344, 346, 348, 350, 352, 354,356, 358, 360, 362, 364, 366, 368, 370, 372, 374, 376, 378, 380, 382,384, 386, 388, 390, 392, 394, 396, 398, 400, 402, 404, 406, 408, 410,412, 414, 416, 418, 420, 422, 424, 426, 428, 430, 432, 434, 436, 438,440, 442, 444, 446, 448, 450, 452, 454, 456, 458, 460, 462, 464, 466,468, 470, 472, 474, 476, 478, 480, 482, 484, 486, 488, 490, 492, 494,496, 498, 500, 502, 504, 506, 508, 510, 512, 514, 516, 518, 520, 522,524, 526, 528, 530, 532, 534, 536, 538, 540, 542, 544, 546, 548, 550,552, 554, 556, 558, 560, 562, 564, 566, 568, 570, 572, 574, 576, 578,580, 582, 584, 586, 588, 590, 592, 594, 596, 598, 600, 602, 604, 606,608, 610, 612, 614, 616, 618, 620, 622, 624, 626, 628, 630, 632, 634,636, 638, 640, 642, 644, 646, 648, 650, 652, 654, 656, 658, 660, 662,664, 666, 668, 670, 672, 674, 676, 678, 680, 682, 684, 686, 688, 690,692, 694, 696, 698, 700, 702, 704, 706, 708, 710, 712, 714, 716, 718,720, 722, 724, 726, 728, 730, 732, 734, 736, 738, 740, 742, 744, 746,748, 750, 752, 754, 770, 772, 774, 776, 778, 780, 782, 784, 786, 788,790, 792, 794, 796, 798, 800, 802, 804, 806, 808, 810, 812, 814, 816,818, 820, 822, 824, 826, 828, 830, 832, 834, 836, 838, 840, 842, 844,846, 848, 850, 852, 854, 856, 858, 860, 862, 864, 866, 868, 870, 872,874, 876, 878, 880, 882, 884, 886, 888, 890, 892, 894, 896, 898, 900,902, 904, 906, 908, 910, 912, 914, 916, 918, 920, 922, 924, 926, 928,930, 932, 934, 936, 938, 940, 942, 944, 946, 948, 950, 952, 954, 956,958, 960, 962, 964, 966, 968, 970, 972, 974, 976, 978, 980, 982, 984,986, 988, 990, 992, 994, 996, 998, 1000, 1002, 1004, 1006, 1008, 1010,1012, 1014, 1016, 1018, 1020, 1022, 1024, 1026, 1028, 1030, 1032, 1034,1036, 1038, 1040, 1042, 1044, 1046, 1048, 1050, 1052, 1054, 1056, 1058,1060, 1062, 1064, 1066, 1068, 1070, 1072, 1074, 1076, 1078, 1290, 1292,1294, 2596, 2598, 2600, 2602, 2604, 2606, 2608, 2610, 2612, 2614, 2616,2618, 2620, 2622, 2624, 2626, 2628, 2630, 2632, 2634, 2636, 2638, 2640,2642, 2644, 2646, 2648, 2650, 2652, 2654, 2656, 2658, 2660, 2662, 2664,2666, 2668, 2670, 2672, 2674, 2676, 2678, 2680, 2682, 2684, 2686, 2688,2690, 2692, 2694, 2696, 2698, 2700, 2702, 2704, 2706, 2708, 2710, 2712,2714, 2716, 2718, 2720, 2722, 2724, 2726, 2728, 2730, 2732, 2734, 2736,2738, 2740, 2742, 2744, 2746, 2748, 2750, 2752, 2754, 2756, 2758, 2760,2762, 2764, 2766, 2768, 2770, 2772, 2774, 2776, 2778, 2780, 2782, 2784,2786, 2788, 2790, 2792, 2794, 2796, 2798, 2800, 2802, 2804, 2806, 2808,2810, 2812, 2814, 2816, 2818, 2820, 2822, 2824, 2826, 2828, 2830, 2832,2834, 2836, 2838, 2840, 2842, 2844, 2846, 2848, 2850, 2852, 2854, 2856,2858, 2860, 2862, 2864, 2866, 2868, 2870, 2872, 2874, 2876, 2878, 2880,2882, 2884, 2886, 2888, 2890, 2892, 2894, 2896, 2898, 2900, 2902, 2904,2906, 2908, 2910, 2912, 2914, 2916, 2918, 2920, 2922, 2924, 2926, 2928,2830, 2932, 2934, 2936, 2938, 2940, 2942, 2944, 2946, 2948, 2950, 2952,2954, 2956, 2958, 2960, 2962, 2964, 2966, 2968, 2970, 2972, 2974, 2976,2978, 2980, 2982, 2984, 2986, 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7032,7034, 7036, 7038, 7040, 7042, 7044, 7046, 7048, 7050, 7052, 7054, 7056,7058, 7060, 7062, 7064, 7066, 7068, 7070, 7072, 7074, 7076, 7078, 7080,7082, 7084, 7086, 7088, 7090, 7092, 7094, 7096, 7098, 7100, 7102, 7104,7106, 7108, 7110, 7112, 7114, 7116, 7118, 7120, 7122, 7124, 7126, 7128,7130, 7132, 7134, 7136, 7138, 7140, 7142, 7144, 7146, 7148, 7150, 7152,7154, 7156, 7158, 7160, 7162, 7164, 7166, 7168, 7170, 7172, 7174, 7176,7178, 7180, 7182, 7184, 7186, 7188, 7190, 7192, 7194, 7196, 7198, 7200,7202, 7204, 7206, 7208, 7210, 7212, 7214, 7216, 7218, 7220, 7222, 7224,7226, 7228, 7230, 7232, 7234, 7236, 7238, 7240, 7242, 7244, 7246, 7248,7250, 7252, 7254, 7256, 7258, 7260, 7262, 7264, 7266, 7268, 7270, 7272,7274, 7276, 7278, 7280, 7282, 7284, 7286, 7288, 7290, 7292, 7294, 7296,7298, 7300, 7302, 7304, 7306, 7308, 7310, 7312, 7314, 7316, 7318, 7320,7322, 7324, 7326, 7328, 7330, 7332, 7334, 7336, 7338, 7340, 7342, 7344,7346, 7348, 7350, 7352, 7354, 7356, 7358, 7360, 7362, 7364, 7366, 7368,7370, 7372, 7374, 7376, 7378, 7380, 7382, 7384, 7386, 7388, 7390, 7392,7394, 7396, 7398, 7400, 7402, 7404, 7406, 7408, 7410, 7412, 7414, 7416,7418, 7420, 7422, 7424, 7426, 7428, 7430, 7432, 7434, 7436, 7766, 7768,7770, 7772, 7774, 7776, 7778, 7780, 7782, 7784, 7786, 7788, 7790, 7792,7794, 7796, 7798, 7800, 7812, 7814, 7816, 7818, 7820, 7822, 7824, 7826,7828, 7830, 7832, 7834, 7836, 7838, 7840, 7842, 7844, 7846, 7848, 7850,7852, 7854, 7856, 7858, 7860, 7862, 7864, 7866, 7868, 7870, 7872, 7874,7876, 7878, 7880, 7882, 7884, 7886, 7888, 7890, 7892, 7894, 7896, 7898,7900, 7902, 7904, 7906, 7908, 7910, 7912, 7914, 7916, 7918, 7920, 7922,7924, 7926, 7928, 7930, 7932, 7934, 7936, 7938, 7940, 7942, 7944, 7946,7948, 7950, 7952, 7954, 7956, 7958, 7960, 7962, 7964, 7966, 7968, 7970,7972, 7974, 7976, 7978, 7980, 7982, 7984, 7986, 7988, 7990, 7992, 7994,7996, 7998, 8000, 8002, 8004, 8006, 8008, 8010, 8012, 8014, 8016, 8018,8020, 8022, 8024, 8026, 8028, 8030, 8032, 8034, 8036, 8038, 8040, 8042,8044, 8046, 8048, 8050, 8052, 8054, 8056, 8058, 8060, 8062, 8064, 8066,8068, 8070, 8072, 8074, 8076, 8078, 8080, 8082, 8084, 8086, 8088, 8090,8092, 8094, 8096, 8098, 8100, 8102, 8104, 8106, 8108, 8110, 8112, 8114,8116, 8118, 8120, 8122, 8124, 8126, 8128, 8130, 8132, 8134, 8136, 8138,8140, 8142, 8144, 8146, 8148, 8150, 8152, 8154, 8156, 8158, 8160, 8162,8164, 8166, 8168, 8170, 8172, 8174, 8176, 8178, 8180, 8182, 8184, 8186,8188, 8190, 8192, 8194, 8196, 8198, 8200, 8202, 8204, 8206, 8208, 8210,8212, 8214, 8216, 8218, 8220, 8222, 8224, 8226, 8228, 8230, 8232, 8234,8236, 8238, 8240, 8242, 8244, 8246, 8248, 8250, 8252, 8254, 8256, 8258,8260, 8262, 8264, 8266, 8268, 8270, 8272, 8274, 8276, 8278, 8280, 8282,8284, 8286, 8288, 8290, 8292, 8294, 8296, 8298, 8300, 8302, 8304, 8306,8308, 8310, 8312, 8314, 8316, 8318, 8320, 8322, 8324, 8326, 8328, 8330,8332, 8334, 8336, 8338, 8340, 8342, 8344, 8346, 8348, 8350, 8352, 8354,8356, 8358, 8360, 8362, 8364, 8366, 8368, 8482, 8484, 8486, 8488, 8490,8492, 8494, 8496, 8498, 8500, 8502, 8504, 8506, 8508, 8510, 8512, 8514,8516, 8518, 8520, 8522, 8524, 8526, 8528, 8530, 8532, 8534, 8536, 8538,8540, 8542, 8544, 8546, 8548, 8550, 8552, 8554, 8556, 8558, 8560, 8562,8564, 8566, 8568, 8570, 8572, 8574, 8576, 8578, 8580, 8582, 8584, 8586,8588, 8590, 8592, 8594, 8596, 8598, 8600, 8602, 8604, 8606, 8608, 8610,8612, 8614, 8616, 8618, 8620, 8622, 8624, 8626, 8628, 8630, 8632, 8634,8636, 8638, 8640, 8642, 8644, 8646, 8648, 8650, 8652, 8654, 8656, 8658,8660, 8662, 8664, 8666, 8668, 8670, 8672, 8674, 8676, 8678, 8680, 8682,8684, 8686, 8688, 8690, 8692, 8694, 8696, 8698, 8700, 8702, 8704, 8706,8708, 8710, 8712, 8714, 8716, 8718, 8720, 8722, 8724, 8726, 8728, 8730,8732, 8734, 8736, 8738, 8740, 8742, 8744, 8746, 8748, 8750, 8752, 8754,8756, 8758, 8760, 8762, 8764, 8766, 8768, 8770, 8772, 8774, 8776, 8778,8780, 8782, 8784, 8786, 8788, 8790, 8792, 8794, 8796, 9108, 9110, 9112,9114, 9116, 9118, 9120, 9122, 9124, 9126, 9128, 9130, 9132, 9134, 9136,9138, 9140, 9142, 9144, 9146, 9148, 9150, 9152, 9154, 9156, 9158, 9160,9162, 9164, 9166, 9168, 9170, 9172, 9174, 9176, 9178, 9180, 9182, 9184,9186, 9188, 9190, 9192, 9194, 9196, 9198, 9200, 9202, 9204, 9206, 9208,9210, 9212, 9214, 9216, 9218, 9220, 9222, 9224, 9226, 9228, 9230, 9232,9234, 9236, 9238, and/or 9240. In some additional embodiments, thepresent invention further provides engineered sucrose synthasescomprising polypeptide sequences that have at least 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to SEQ IDNOS:1080, 1082, 1084, 1086, 1088, 1090, 1092, 1094, 1096, 1098, 1100,1102, 1104, 1106, 1108, 1110, 1112, 1114, 1116, 1118, 1120, 1122, 1124,1126, 1128, 1130, 1132, 1134, 1136, 1138, 1140, 1142, 1144, 1146, 1148,1150, 1152, 1154, 1156, 1158, 1160, 1162, 1164, 1166, 1168, 1170, 1172,1174, 1176, 1178, 1180, 1182, 1184, 1186, 1188, 1190, 1192, 1194, 1196,1198, 1200, 1202, 1204, 1206, 1208, 1210, 1212, 1214, 1216, 1218, 1220,1222, 1224, 1226, 1228, 1230, 1232, 1234, 1236, 1238, 1240, 1242, 1244,1246, 1248, 1250, 1252, 1254, 1256, 1258, 1260, 1262, 1264, 1266, 1268,1270, 1272, 1274, 1276, 1278, 1280, 1282, 1284, 1286, 1288, 1296, 1298,1300, 1302, 1304, 1306, 1308, 1310, 1312, 1314, 1316, 1318, 1320, 1322,1324, 1326, 1328, 1330, 1332, 1334, 1336, 1338, 1340, 1342, 1344, 1346,1348, 1350, 1352, 1354, 1356, 1358, 1360, 1362, 1364, 1366, 1368, 1370,1372, 1374, 1376, 1378, 1380, 1382, 1384, 1386, 1388, 1390, 1392, 1394,1396, 1398, 1400, 1402, 1404, 1406, 1408, 1410, 1412, 1414, 1416, 1418,1420, 1422, 1424, 1426, 1428, 1430, 1432, 1434, 1436, 1438, 1440, 1442,1444, 1446, 1448, 1450, 1452, 1454, 1456, 1458, 1460, 1462, 1464, 1466,1468, 1470, 1472, 1474, 1476, 1478, 1480, 1482, 1484, 1486, 1488, 1490,1492, 1494, 1496, 1498, 1500, 1502, 1504, 1506, 1508, 1510, 1512, 1514,1516, 1518, 1520, 1522, 1524, 1526, 1528, 1530, 1532, 1534, 1536, 1538,1540, 1542, 1544, 1546, 1548, 1550, 1552, 1554, 1556, 1558, 1560, 1562,1564, 1566, 1568, 1570, 1572, 1574, 1576, 1578, 1580, 1582, 1584, 1586,1588, 1590, 1592, 1594, 1596, 1598, 1600, 1602, 1604, 1606, 1608, 1610,1612, 1614, 1616, 1618, 1620, 1622, 1624, 1626, 1628, 1630, 1632, 1634,1636, 1638, 1640, 1642, 1644, 1646, 1648, 1650, 1652, 1654, 1656, 1658,1660, 1662, 1664, 1666, 1668, 1670, 1672, 1674, 1676, 1678, 1680, 1682,1684, 1686, 1688, 1690, 1692, 1694, 1696, 1698, 1700, 1702, 1704, 1706,1708, 1710, 1712, 1714, 1716, 1718, 1720, 1722, 1724, 1726, 1728, 1730,1732, 1734, 1736, 1738, 1740, 1742, 1744, 1746, 1748, 1750, 1752, 1754,1756, 1758, 1760, 1762, 1764, 1766, 1768, 1770, 1772, 1774, 1776, 1778,1780, 1782, 1784, 1786, 1788, 1790, 1792, 1794, 1796, 1798, 1800, 1802,1804, 1806, 1808, 1810, 1812, 1814, 1816, 1818, 1820, 1822, 1824, 1826,1828, 1830, 1832, 1834, 1836, 1838, 1840, 1842, 1844, 1846, 1848, 1850,1852, 1854, 1856, 1858, 1860, 1862, 1864, 1866, 1868, 1870, 1872, 1874,1876, 1878, 1880, 1882, 1884, 1886, 1888, 1890, 1892, 1894, 1896, 1898,1900, 1902, 1904, 1906, 1908, 1910, 1912, 1914, 1916, 1918, 1920, 1922,1924, 1926, 1928, 1930, 1932, 1934, 1936, 1938, 1940, 1942, 1944, 1946,1948, 1950, 1952, 1954, 1956, 1958, 1960, 1962, 1964, 1966, 1968, 1970,1972, 1974, 1976, 1978, 1980, 1982, 1984, 1986, 1988, 1990, 1992, 1994,1996, 1998, 2000, 2002, 2004, 2006, 2008, 2010, 2012, 2014, 2016, 2018,2020, 2022, 2024, 2026, 2028, 2030, 2032, 2034, 2036, 2038, 2040, 2042,2044, 2046, 2048, 2050, 2052, 2054, 2056, 2058, 2060, 2062, 2064, 2066,2068, 2070, 2072, 2074, 2076, 2078, 2080, 2082, 2084, 2086, 2088, 2090,2092, 2094, 2096, 2098, 2100, 2102, 2104, 2106, 2108, 2110, 2112, 2114,2116, 2118, 2120, 2122, 2124, 2126, 2128, 2130, 2132, 2134, 2136, 2138,2140, 2142, 2144, 2146, 2148, 2150, 2152, 2154, 2156, 2158, 2160, 2162,2164, 2166, 2168, 2170, 2172, 2174, 2176, 2178, 2180, 2182, 2184, 2186,2188, 2190, 2192, 2194, 2196, 2198, 2200, 2202, 2204, 2206, 2208, 2210,2212, 2214, 2216, 2218, 2220, 2222, 2224, 2226, 2228, 2230, 2232, 2234,2236, 2238, 2240, 2242, 2244, 2246, 2248, 2250, 2252, 2254, 2256, 2258,2260, 2262, 2264, 2266, 2268, 2270, 2272, 2274, 2276, 2278, 2280, 2282,2284, 2286, 2288, 2290, 2292, 2294, 2296, 2298, 2300, 2302, 2304, 2306,2308, 2310, 2312, 2314, 2316, 2318, 2320, 2322, 2324, 2326, 2328, 2330,2332, 2334, 2336, 2338, 2340, 2342, 2344, 2346, 2348, 2350, 2352, 2354,2356, 2358, 2360, 2362, 2364, 2366, 2368, 2370, 2372, 2374, 2376, 2378,2380, 2382, 2384, 2386, 2388, 2390, 2392, 2394, 2396, 2398, 2400, 2402,2404, 2406, 2408, 2410, 2412, 2414, 2416, 2418, 2420, 2422, 2424, 2426,2428, 2430, 2432, 2434, 2436, 2438, 2440, 2442, 2444, 2446, 2448, 2450,2452, 2454, 2456, 2458, 2460, 2462, 2464, 2466, 2468, 2470, 2472, 2474,2476, 2478, 2480, 2482, 2484, 2486, 2488, 2490, 2492, 2494, 2496, 2498,2500, 2502, 2504, 2506, 2508, 2510, 2512, 2514, 2516, 2518, 2520, 2522,2524, 2526, 2528, 2530, 2532, 2534, 2536, 2538, 2540, 2542, 2544, 2546,2548, 2550, 2552, 2554, 2556, 2558, 2560, 2562, 2564, 2566, 2568, 2570,2572, 2574, 2576, 2578, 2580, 2582, 2584, 2586, 2588, 2590, 2592, 2594,7438, 7440, 7442, 7444, 7446, 7448, 7450, 7452, 7454, 7456, 7458, 7460,7462, 7464, 7466, 7468, 7470, 7472, 7474, 7476, 7478, 7480, 7482, 7484,7486, 7488, 7490, 7492, 7494, 7496, 7498, 7500, 7502, 7504, 7506, 7508,7510, 7512, 7514, 7516, 7518, 7520, 7522, 7524, 7526, 7528, 7530, 7532,7534, 7536, 7538, 7540, 7542, 7544, 7546, 7548, 7550, 7552, 7554, 7556,7558, 7560, 7562, 7564, 7566, 7568, 7570, 7572, 7574, 7576, 7578, 7580,7582, 7584, 7586, 7588, 7590, 7592, 7594, 7596, 7598, 7600, 7602, 7604,7606, 7608, 7610, 7612, 7614, 7616, 7618, 7620, 7622, 7624, 7626, 7628,7630, 7632, 7634, 7636, 7638, 7640, 7642, 7644, 7646, 7648, 7650, 7652,7654, 7656, 7658, 7660, 7662, 7664, 7666, 7668, 7670, 7672, 7674, 7676,7678, 7680, 7682, 7684, 7686, 7688, 7690, 7692, 7694, 7696, 7698, 7700,7702, 7704, 7706, 7708, 7710, 7712, 7714, 7716, 7718, 7720, 7722, 7724,7726, 7728, 7730, 7732, 7734, 7736, 7738, 7740, 7742, 7744, 7746, 7748,7750, 7752, 7754, 7756, 7758, 7760, 7762, 7764, 8370, 8372, 8374, 8376,8378, 8380, 8382, 8384, 8386, 8388, 8390, 8392, 8394, 8396, 8398, 8400,8402, 8404, 8406, 8408, 8410, 8412, 8414, 8416, 8418, 8420, 8422, 8424,8426, 8428, 8430, 8432, 8434, 8436, 8438, 8440, 8442, 8444, 8446, 8448,8450, 8452, 8454, 8456, 8458, 8460, 8462, 8464, 8466, 8468, 8470, 8472,8474, 8476, 8478, 8480, 8798, 8800, 8802, 8804, 8806, 8808, 8810, 8812,8814, 8816, 8818, 8820, 8822, 8824, 8826, 8828, 8830, 8832, 8834, 8836,8838, 8840, 8842, 8844, 8846, 8848, 8850, 8852, 8854, 8856, 8858, 8860,8862, 8864, 8866, 8868, 8870, 8872, 8874, 8876, 8878, 8880, 8882, 8884,8886, 8888, 8890, 8892, 8894, 8896, 8898, 8900, 8902, 8904, 8906, 8908,8910, 8912, 8914, 8916, 8918, 8920, 8922, 8924, 8926, 8928, 8930, 8932,8934, 8936, 8938, 8940, 8942, 8944, 8946, 8948, 8950, 8952, 8954, 8956,8958, 8960, 8962, 8964, 8966, 8968, 8970, 8972, 8974, 8976, 8978, 8980,8982, 8984, 8986, 8988, 8990, 8992, 8994, 8996, 8998, 9000, 9002, 9004,9006, 9008, 9010, 9012, 9014, 9016, 9018, 9020, 9022, 9024, 9026, 9028,9030, 9032, 9034, 9036, 9038, 9040, 9042, 9044, 9046, 9048, 9050, 9052,9054, 9056, 9058, 9060, 9062, 9064, 9066, 9068, 9070, 9072, 9074, 9076,9078, 9080, 9082, 9084, 9086, 9088, 9090, 9092, 9094, 9096, 9098, 9100,9102, 9104, and 9106.

In some embodiments, additional reaction components or additionaltechniques are utilized to supplement the reaction conditions. In someembodiments, these include taking measures to stabilize or preventinactivation of the enzyme, reduce product inhibition, shift reactionequilibrium to glucosylated product formation.

In some further embodiments, any of the above described process for theconversion of substrate compound to product compound can furthercomprise one or more steps selected from: extraction; isolation;purification; and crystallization, filtration, or lyophilization ofproduct compound. Methods, techniques, and protocols for extracting,isolating, purifying, and/or crystallizing the glucosylated product frombiocatalytic reaction mixtures produced by the above disclosed processesare known to the ordinary artisan and/or accessed through routineexperimentation. Additionally, illustrative methods are provided in theExamples below.

Engineered Sucrose Synthase Polypeptides

The present invention provides engineered sucrose synthase (SuS)polypeptides, polynucleotides encoding the polypeptides, methods ofpreparing the polypeptides, and methods for using the polypeptides.Where the description relates to polypeptides, it is to be understoodthat it also describes the polynucleotides encoding the polypeptides. Insome embodiments, the present invention provides engineered,non-naturally occurring SuS enzymes with improved properties as comparedto wild-type SuS enzymes. Any suitable reaction conditions find use inthe present invention. In some embodiments, methods are used to analyzethe improved properties of the engineered polypeptides to carry out thesynthase reaction. In some embodiments, the reaction conditions aremodified with regard to concentrations or amounts of engineered SuS,substrate(s), buffer(s), solvent(s), pH, conditions includingtemperature and reaction time, and/or conditions with the engineered SuSpolypeptide immobilized on a solid support, as further described belowand in the Examples.

In some embodiments, the engineered SuS polypeptides described hereinhave improved properties as compared to wild-type SuS enzymes such as inthe reactions described herein. In some embodiments, the engineered SuSenzymes comprise amino acid sequences having one or more residuedifferences as compared to SEQ ID NO: 74, 1080, 1158, 1222, 1392, 1456,1582, 1764, 1804, 1840, 2064, 2432, 2510, 7506, and/or 8420.

In some embodiments, the present invention provides engineered SuSenzymes, wherein the polypeptide of the SuS enzymes comprise at least60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to SEQ IDNO:1080, 1082, 1084, 1086, 1088, 1090, 1092, 1094, 1096, 1098, 1100,1102, 1104, 1106, 1108, 1110, 1112, 1114, 1116, 1118, 1120, 1122, 1124,1126, 1128, 1130, 1132, 1134, 1136, 1138, 1140, 1142, 1144, 1146, 1148,1150, 1152, 1154, 1156, 1158, 1160, 1162, 1164, 1166, 1168, 1170, 1172,1174, 1176, 1178, 1180, 1182, 1184, 1186, 1188, 1190, 1192, 1194, 1196,1198, 1200, 1202, 1204, 1206, 1208, 1210, 1212, 1214, 1216, 1218, 1220,1222, 1224, 1226, 1228, 1230, 1232, 1234, 1236, 1238, 1240, 1242, 1244,1246, 1248, 1250, 1252, 1254, 1256, 1258, 1260, 1262, 1264, 1266, 1268,1270, 1272, 1274, 1276, 1278, 1280, 1282, 1284, 1286, 1288, 1296, 1298,1300, 1302, 1304, 1306, 1308, 1310, 1312, 1314, 1316, 1318, 1320, 1322,1324, 1326, 1328, 1330, 1332, 1334, 1336, 1338, 1340, 1342, 1344, 1346,1348, 1350, 1352, 1354, 1356, 1358, 1360, 1362, 1364, 1366, 1368, 1370,1372, 1374, 1376, 1378, 1380, 1382, 1384, 1386, 1388, 1390, 1392, 1394,1396, 1398, 1400, 1402, 1404, 1406, 1408, 1410, 1412, 1414, 1416, 1418,1420, 1422, 1424, 1426, 1428, 1430, 1432, 1434, 1436, 1438, 1440, 1442,1444, 1446, 1448, 1450, 1452, 1454, 1456, 1458, 1460, 1462, 1464, 1466,1468, 1470, 1472, 1474, 1476, 1478, 1480, 1482, 1484, 1486, 1488, 1490,1492, 1494, 1496, 1498, 1500, 1502, 1504, 1506, 1508, 1510, 1512, 1514,1516, 1518, 1520, 1522, 1524, 1526, 1528, 1530, 1532, 1534, 1536, 1538,1540, 1542, 1544, 1546, 1548, 1550, 1552, 1554, 1556, 1558, 1560, 1562,1564, 1566, 1568, 1570, 1572, 1574, 1576, 1578, 1580, 1582, 1584, 1586,1588, 1590, 1592, 1594, 1596, 1598, 1600, 1602, 1604, 1606, 1608, 1610,1612, 1614, 1616, 1618, 1620, 1622, 1624, 1626, 1628, 1630, 1632, 1634,1636, 1638, 1640, 1642, 1644, 1646, 1648, 1650, 1652, 1654, 1656, 1658,1660, 1662, 1664, 1666, 1668, 1670, 1672, 1674, 1676, 1678, 1680, 1682,1684, 1686, 1688, 1690, 1692, 1694, 1696, 1698, 1700, 1702, 1704, 1706,1708, 1710, 1712, 1714, 1716, 1718, 1720, 1722, 1724, 1726, 1728, 1730,1732, 1734, 1736, 1738, 1740, 1742, 1744, 1746, 1748, 1750, 1752, 1754,1756, 1758, 1760, 1762, 1764, 1766, 1768, 1770, 1772, 1774, 1776, 1778,1780, 1782, 1784, 1786, 1788, 1790, 1792, 1794, 1796, 1798, 1800, 1802,1804, 1806, 1808, 1810, 1812, 1814, 1816, 1818, 1820, 1822, 1824, 1826,1828, 1830, 1832, 1834, 1836, 1838, 1840, 1842, 1844, 1846, 1848, 1850,1852, 1854, 1856, 1858, 1860, 1862, 1864, 1866, 1868, 1870, 1872, 1874,1876, 1878, 1880, 1882, 1884, 1886, 1888, 1890, 1892, 1894, 1896, 1898,1900, 1902, 1904, 1906, 1908, 1910, 1912, 1914, 1916, 1918, 1920, 1922,1924, 1926, 1928, 1930, 1932, 1934, 1936, 1938, 1940, 1942, 1944, 1946,1948, 1950, 1952, 1954, 1956, 1958, 1960, 1962, 1964, 1966, 1968, 1970,1972, 1974, 1976, 1978, 1980, 1982, 1984, 1986, 1988, 1990, 1992, 1994,1996, 1998, 2000, 2002, 2004, 2006, 2008, 2010, 2012, 2014, 2016, 2018,2020, 2022, 2024, 2026, 2028, 2030, 2032, 2034, 2036, 2038, 2040, 2042,2044, 2046, 2048, 2050, 2052, 2054, 2056, 2058, 2060, 2062, 2064, 2066,2068, 2070, 2072, 2074, 2076, 2078, 2080, 2082, 2084, 2086, 2088, 2090,2092, 2094, 2096, 2098, 2100, 2102, 2104, 2106, 2108, 2110, 2112, 2114,2116, 2118, 2120, 2122, 2124, 2126, 2128, 2130, 2132, 2134, 2136, 2138,2140, 2142, 2144, 2146, 2148, 2150, 2152, 2154, 2156, 2158, 2160, 2162,2164, 2166, 2168, 2170, 2172, 2174, 2176, 2178, 2180, 2182, 2184, 2186,2188, 2190, 2192, 2194, 2196, 2198, 2200, 2202, 2204, 2206, 2208, 2210,2212, 2214, 2216, 2218, 2220, 2222, 2224, 2226, 2228, 2230, 2232, 2234,2236, 2238, 2240, 2242, 2244, 2246, 2248, 2250, 2252, 2254, 2256, 2258,2260, 2262, 2264, 2266, 2268, 2270, 2272, 2274, 2276, 2278, 2280, 2282,2284, 2286, 2288, 2290, 2292, 2294, 2296, 2298, 2300, 2302, 2304, 2306,2308, 2310, 2312, 2314, 2316, 2318, 2320, 2322, 2324, 2326, 2328, 2330,2332, 2334, 2336, 2338, 2340, 2342, 2344, 2346, 2348, 2350, 2352, 2354,2356, 2358, 2360, 2362, 2364, 2366, 2368, 2370, 2372, 2374, 2376, 2378,2380, 2382, 2384, 2386, 2388, 2390, 2392, 2394, 2396, 2398, 2400, 2402,2404, 2406, 2408, 2410, 2412, 2414, 2416, 2418, 2420, 2422, 2424, 2426,2428, 2430, 2432, 2434, 2436, 2438, 2440, 2442, 2444, 2446, 2448, 2450,2452, 2454, 2456, 2458, 2460, 2462, 2464, 2466, 2468, 2470, 2472, 2474,2476, 2478, 2480, 2482, 2484, 2486, 2488, 2490, 2492, 2494, 2496, 2498,2500, 2502, 2504, 2506, 2508, 2510, 2512, 2514, 2516, 2518, 2520, 2522,2524, 2526, 2528, 2530, 2532, 2534, 2536, 2538, 2540, 2542, 2544, 2546,2548, 2550, 2552, 2554, 2556, 2558, 2560, 2562, 2564, 2566, 2568, 2570,2572, 2574, 2576, 2578, 2580, 2582, 2584, 2586, 2588, 2590, 2592, 2594,7438, 7440, 7442, 7444, 7446, 7448, 7450, 7452, 7454, 7456, 7458, 7460,7462, 7464, 7466, 7468, 7470, 7472, 7474, 7476, 7478, 7480, 7482, 7484,7486, 7488, 7490, 7492, 7494, 7496, 7498, 7500, 7502, 7504, 7506, 7508,7510, 7512, 7514, 7516, 7518, 7520, 7522, 7524, 7526, 7528, 7530, 7532,7534, 7536, 7538, 7540, 7542, 7544, 7546, 7548, 7550, 7552, 7554, 7556,7558, 7560, 7562, 7564, 7566, 7568, 7570, 7572, 7574, 7576, 7578, 7580,7582, 7584, 7586, 7588, 7590, 7592, 7594, 7596, 7598, 7600, 7602, 7604,7606, 7608, 7610, 7612, 7614, 7616, 7618, 7620, 7622, 7624, 7626, 7628,7630, 7632, 7634, 7636, 7638, 7640, 7642, 7644, 7646, 7648, 7650, 7652,7654, 7656, 7658, 7660, 7662, 7664, 7666, 7668, 7670, 7672, 7674, 7676,7678, 7680, 7682, 7684, 7686, 7688, 7690, 7692, 7694, 7696, 7698, 7700,7702, 7704, 7706, 7708, 7710, 7712, 7714, 7716, 7718, 7720, 7722, 7724,7726, 7728, 7730, 7732, 7734, 7736, 7738, 7740, 7742, 7744, 7746, 7748,7750, 7752, 7754, 7756, 7758, 7760, 7762, 7764, 8370, 8372, 8374, 8376,8378, 8380, 8382, 8384, 8386, 8388, 8390, 8392, 8394, 8396, 8398, 8400,8402, 8404, 8406, 8408, 8410, 8412, 8414, 8416, 8418, 8420, 8422, 8424,8426, 8428, 8430, 8432, 8434, 8436, 8438, 8440, 8442, 8444, 8446, 8448,8450, 8452, 8454, 8456, 8458, 8460, 8462, 8464, 8466, 8468, 8470, 8472,8474, 8476, 8478, 8480, 8798, 8800, 8802, 8804, 8806, 8808, 8810, 8812,8814, 8816, 8818, 8820, 8822, 8824, 8826, 8828, 8830, 8832, 8834, 8836,8838, 8840, 8842, 8844, 8846, 8848, 8850, 8852, 8854, 8856, 8858, 8860,8862, 8864, 8866, 8868, 8870, 8872, 8874, 8876, 8878, 8880, 8882, 8884,8886, 8888, 8890, 8892, 8894, 8896, 8898, 8900, 8902, 8904, 8906, 8908,8910, 8912, 8914, 8916, 8918, 8920, 8922, 8924, 8926, 8928, 8930, 8932,8934, 8936, 8938, 8940, 8942, 8944, 8946, 8948, 8950, 8952, 8954, 8956,8958, 8960, 8962, 8964, 8966, 8968, 8970, 8972, 8974, 8976, 8978, 8980,8982, 8984, 8986, 8988, 8990, 8992, 8994, 8996, 8998, 9000, 9002, 9004,9006, 9008, 9010, 9012, 9014, 9016, 9018, 9020, 9022, 9024, 9026, 9028,9030, 9032, 9034, 9036, 9038, 9040, 9042, 9044, 9046, 9048, 9050, 9052,9054, 9056, 9058, 9060, 9062, 9064, 9066, 9068, 9070, 9072, 9074, 9076,9078, 9080, 9082, 9084, 9086, 9088, 9090, 9092, 9094, 9096, 9098, 9100,9102, 9104, and 9106

In some embodiments, additional reaction components or additionaltechniques are utilized to supplement the reaction conditions. In someembodiments, these include taking measures to stabilize or preventinactivation of the enzyme, reduce product inhibition, shift reactionequilibrium to glucosylated product formation.

In some further embodiments, any of the above described process for theconversion of substrate compound to product compound can furthercomprise one or more steps selected from: extraction, isolation,purification, crystallization, filtration, and/or lyophilization ofproduct compound(s). Methods, techniques, and protocols for extracting,isolating, purifying, and/or crystallizing the product(s) (e.g.,rebaudiosides) from biocatalytic reaction mixtures produced by theprocesses provided herein are known to the ordinary artisan and/oraccessed through routine experimentation. Additionally, illustrativemethods are provided in the Examples below.

Polynucleotides Encoding Engineered Polypeptides, Expression Vectors andHost Cells

The present invention provides polynucleotides encoding the engineeredenzyme polypeptides described herein. In some embodiments, thepolynucleotides are operatively linked to one or more heterologousregulatory sequences that control gene expression to create arecombinant polynucleotide capable of expressing the polypeptide. Insome embodiments, expression constructs containing at least oneheterologous polynucleotide encoding the engineered enzymepolypeptide(s) is introduced into appropriate host cells to express thecorresponding enzyme polypeptide(s).

As will be apparent to the skilled artisan, availability of a proteinsequence and the knowledge of the codons corresponding to the variousamino acids provide a description of all the polynucleotides capable ofencoding the subject polypeptides. The degeneracy of the genetic code,where the same amino acids are encoded by alternative or synonymouscodons, allows an extremely large number of nucleic acids to be made,all of which encode an engineered enzyme (e.g., GT or SuS) polypeptide.Thus, the present invention provides methods and compositions for theproduction of each and every possible variation of enzymepolynucleotides that could be made that encode the enzyme polypeptidesdescribed herein by selecting combinations based on the possible codonchoices, and all such variations are to be considered specificallydisclosed for any polypeptide described herein, including the amino acidsequences presented in the Examples (e.g., in the various Tables).

In some embodiments, the codons are preferably optimized for utilizationby the chosen host cell for protein production. For example, preferredcodons used in bacteria are typically used for expression in bacteria.Consequently, codon optimized polynucleotides encoding the engineeredenzyme polypeptides contain preferred codons at about 40%, 50%, 60%,70%, 80%, 90%, or greater than 90% of the codon positions in the fulllength coding region.

In some embodiments, the enzyme polynucleotide encodes an engineeredpolypeptide having enzyme activity with the properties disclosed herein,wherein the polypeptide comprises an amino acid sequence having at least60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99% or more identity to a reference sequenceselected from the SEQ ID NOS provided herein, or the amino acid sequenceof any variant (e.g., those provided in the Examples), and one or moreresidue differences as compared to the reference polynucleotide(s), orthe amino acid sequence of any variant as disclosed in the Examples (forexample 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acid residuepositions). In some embodiments, the reference polypeptide sequence isselected from SEQ ID NOS: 4, 8, 32, 232, 348, 548, 562, 696, 758, 770,792, 954, 1002, 1054, 2600, 2718, 2814, 2884, 3016, 3082, 3244, 3346,3502, 3696, 3956, 4256, 4550, 4684, 4838, 4876, 5066, 5290, 5372, 5562,5708, 5976, 6138, 6288, 6468, 6864, 7324, 7388, 7784, and/or 8088. Insome alternative embodiments, the reference polypeptide sequence isselected from SEQ ID NOS: 74, 1080, 1158, 1222, 1392, 1456, 1582, 1764,1804, 1840, 2064, 2432, 2510, 7506, and/or 8420.

In some embodiments, the reference polynucleotide sequence is selectedfrom SEQ ID NOS: 3, 7, 31, 231, 347, 547, 561, 695, 757, 769, 791, 953,1001, 1053, 2599, 2717, 2813, 2883, 3015, 3081, 3243, 3345, 3501, 3695,3955, 4255, 4549, 4683, 4837, 4875, 5065, 5289, 5371, 5561, 5707, 5975,6137, 6287, 6467, 6863, 7323, 7387, 7783, and/or 8087. In someembodiments, the reference polynucleotide sequence is selected from SEQID NOS: 757, 769, 791, 953, 1001, 1053, 2599, 2717, 2813, 2883, 3015,3081, 3243, 3345, 3501, 3695, 3955, 4255, 4549, 7323, and/or 7783, whilein some alternative embodiments, the reference polynucleotide sequenceis selected from SEQ ID NOS: 3, 7, 31, 231, 347, 547, 561, 695, 4683,4837, 4875, 5065, 5289, 5371, 5561, 5707, 5975, 6137, 6287, 6467, 6863,7387, and/or 8087. In still some additional embodiments, the referencepolynucleotide sequence is selected from SEQ ID NOS: 73, 1079, 1157,1221, 1391, 1455, 1581, 1763, 1803, 1839, 2063, 2431, 2509, 7505, and/or8419.

In some embodiments, the engineered polynucleotide encoding at least oneengineered glycosyltransferase provided herein comprises at least 60%,65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99%, or more sequence identity to SEQ ID NO:5, 7, 9,11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45,47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 75, 77, 79, 81, 83, 85,87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117,119, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145,147, 149, 151, 153, 155, 157, 159, 161, 163, 165, 167, 169, 171, 173,175, 177, 179, 181, 183, 185, 187, 189, 191, 193, 195, 197, 199, 201,203, 205, 207, 209, 211, 213, 215, 217, 219, 221, 223, 225, 227, 229,231, 233, 235, 237, 239, 241, 243, 245, 247, 249, 251, 253, 255, 257,259, 261, 263, 265, 267, 269, 271, 273, 275, 277, 279, 281, 283, 285,287, 289, 291, 293, 295, 297, 299, 301, 303, 305, 307, 309, 311, 313,315, 317, 319, 321, 323, 325, 327, 329, 331, 333, 335, 337, 339, 341,343, 345, 347, 349, 351, 353, 355, 357, 359, 361, 363, 365, 367, 369,371, 373, 375, 377, 379, 381, 383, 385, 387, 389, 391, 393, 395, 397,399, 401, 403, 405, 407, 409, 411, 413, 415, 417, 419, 421, 423, 425,427, 429, 431, 433, 435, 437, 439, 441, 443, 445, 447, 449, 451, 453,455, 457, 459, 461, 463, 465, 467, 469, 471, 473, 475, 477, 479, 481,483, 485, 487, 489, 491, 493, 495, 497, 499, 501, 503, 505, 507, 509,511, 513, 515, 517, 519, 521, 523, 525, 527, 529, 531, 533, 535, 537,539, 541, 543, 545, 547, 549, 551, 553, 555, 557, 559, 561, 563, 565,567, 569, 571, 573, 575, 577, 579, 581, 583, 585, 587, 589, 591, 593,595, 597, 599, 601, 603, 605, 607, 609, 611, 613, 615, 617, 619, 621,623, 625, 627, 629, 631, 633, 635, 637, 639, 641, 643, 645, 647, 649,651, 653, 655, 657, 659, 661, 663, 665, 667, 669, 671, 673, 675, 677,679, 681, 683, 685, 687, 689, 691, 693, 695, 697, 699, 701, 703, 705,707, 709, 711, 713, 715, 717, 719, 721, 723, 725, 727, 729, 731, 733,735, 737, 739, 741, 743, 745, 747, 749, 751, 753, 769, 771, 773, 775,777, 779, 781, 783, 785, 787, 789, 791, 793, 795, 797, 799, 801, 803,805, 807, 809, 811, 813, 815, 817, 819, 821, 823, 825, 827, 829, 831,833, 835, 837, 839, 841, 843, 845, 847, 849, 851, 853, 855, 857, 859,861, 863, 865, 867, 869, 871, 873, 875, 877, 879, 881, 883, 885, 887,889, 891, 893, 895, 897, 899, 901, 903, 905, 907, 909, 911, 913, 915,917, 919, 921, 923, 925, 927, 929, 931, 933, 935, 937, 939, 941, 943,945, 947, 949, 951, 953, 955, 957, 959, 961, 963, 965, 967, 969, 971,973, 975, 977, 979, 981, 983, 985, 987, 989, 991, 993, 995, 997, 999,1001, 1003, 1005, 1007, 1009, 1011, 1013, 1015, 1017, 1019, 1021, 1023,1025, 1027, 1029, 1031, 1033, 1035, 1037, 1039, 1041, 1043, 1045, 1047,1049, 1051, 1053, 1055, 1057, 1059, 1061, 1063, 1065, 1067, 1069, 1071,1073, 1075, 1077, 1289, 1291, 1293, 2595, 2597, 2599, 2601, 2603, 2605,2607, 2609, 2611, 2613, 2615, 2617, 2619, 2621, 2623, 2625, 2627, 2629,2631, 2633, 2635, 2637, 2639, 2641, 2643, 2645, 2647, 2649, 2651, 2653,2655, 2657, 2659, 2661, 2663, 2665, 2667, 2669, 2671, 2673, 2675, 2677,2679, 2681, 2683, 2685, 2687, 2689, 2691, 2693, 2695, 2697, 2699, 2701,2703, 2705, 2707, 2709, 2711, 2713, 2715, 2717, 2719, 2721, 2723, 2725,2727, 2729, 2731, 2733, 2735, 2737, 2739, 2741, 2743, 2745, 2747, 2749,2751, 2753, 2755, 2757, 2759, 2761, 2763, 2765, 2767, 2769, 2771, 2773,2775, 2777, 2779, 2781, 2783, 2785, 2787, 2789, 2791, 2793, 2795, 2797,2799, 2801, 2803, 2805, 2807, 2809, 2811, 2813, 2815, 2817, 2819, 2821,2823, 2825, 2827, 2829, 2831, 2833, 2835, 2837, 2839, 2841, 2843, 2845,2847, 2849, 2851, 2823, 2855, 2857, 2859, 2861, 2863, 2865, 2867, 2869,2871, 2873, 2875, 2877, 2879, 2881, 2883, 2885, 2887, 2889, 2891, 2893,2895, 2897, 2899, 2901, 2903, 2905, 2907, 2909, 2911, 2913, 2915, 2917,2919, 2921, 2923, 2925, 2927, 2829, 2931, 2933, 2935, 2937, 2939, 2941,2943, 2945, 2947, 2949, 2951, 2953, 2955, 2957, 2959, 2961, 2963, 2965,2967, 2969, 2971, 2973, 2975, 2977, 2979, 2981, 2983, 2985, 2987, 2989,2991, 2993, 2995, 2997, 2999, 3001, 3003, 3005, 3007, 3009, 3011, 3013,3015, 3017, 3019, 3021, 3023, 3025, 3027, 3029, 3031, 3033, 3035, 3037,3039, 3041, 3043, 3045, 3047, 3049, 3051, 3053, 3055, 3057, 3059, 3061,3063, 3065, 3067, 3069, 3071, 3073, 3075, 3077, 3079, 3081, 3083, 3085,3087, 3089, 3091, 3093, 3095, 3097, 3099, 3101, 3103, 3105, 3107, 3109,3111, 3113, 3115, 3117, 3119, 3121, 3123, 3125, 3127, 3129, 3131, 3133,3135, 3137, 3139, 3141, 3143, 3145, 3147, 3149, 3151, 3153, 3155, 3157,3159, 3161, 3163, 3165, 3167, 3169, 3171, 3173, 3175, 3177, 3179, 3181,3183, 3185, 3187, 3189, 3191, 3193, 3195, 3197, 3199, 3201, 3203, 3205,3207, 3209, 3211, 3213, 3215, 3217, 3219, 3221, 3223, 3225, 3227, 3229,3231, 3233, 3235, 3237, 3239, 3241, 3243, 3245, 3247, 3249, 3251, 3253,3255, 3257, 3259, 3261, 3263, 3265, 3267, 3269, 3271, 3273, 3275, 3277,3279, 3281, 3283, 3285, 3287, 3289, 3291, 3293, 3295, 3297, 3299, 3301,3303, 3305, 3307, 3309, 3311, 3313, 3315, 3317, 3319, 3321, 3323, 3325,3327, 3329, 3331, 3333, 3335, 3337, 3339, 3341, 3343, 3345, 3347, 3349,3351, 3353, 3355, 3357, 3359, 3361, 3363, 3365, 3367, 3369, 3371, 3373,3375, 3377, 3379, 3381, 3383, 3385, 3387, 3389, 3391, 3393, 3395, 3397,3399, 3401, 3403, 3405, 3407, 3409, 3411, 3413, 3415, 3417, 3419, 3421,3423, 3425, 3427, 3429, 3431, 3433, 3435, 3437, 3439, 3441, 3443, 3445,3447, 3449, 3451, 3453, 3455, 3457, 3459, 3461, 3463, 3465, 3467, 3469,3471, 3473, 3475, 3477, 3479, 3481, 3483, 3485, 3487, 3489, 3491, 3493,3495, 3497, 3499, 3501, 3503, 3505, 3507, 3509, 3511, 3513, 3515, 3517,3519, 3521, 3523, 3525, 3527, 3529, 3531, 3533, 3535, 3537, 3539, 3541,3543, 3545, 3547, 3549, 3551, 3553, 3555, 3557, 3559, 3561, 3563, 3565,3567, 3569, 3571, 3573, 3575, 3577, 3579, 3581, 3583, 3585, 3587, 3589,3591, 3593, 3595, 3597, 3599, 3601, 3603, 3605, 3607, 3609, 3611, 3613,3615, 3617, 3619, 3621, 3623, 3625, 3627, 3629, 3631, 3633, 3635, 3637,3639, 3641, 3643, 3645, 3647, 3649, 3651, 3653, 3655, 3657, 3659, 3661,3663, 3665, 3667, 3669, 3671, 3673, 3675, 3677, 3679, 3681, 3683, 3685,3687, 3689, 3691, 3693, 3695, 3697, 3699, 3791, 3703, 3705, 3707, 3709,3711, 3713, 3715, 3717, 3719, 3721, 3723, 3725, 3727, 3729, 3731, 3733,3735, 3737, 3739, 3741, 3743, 3745, 3747, 3749, 3751, 3753, 3755, 3757,3759, 3761, 3763, 3765, 3767, 3769, 3771, 3773, 3775, 3777, 3779, 3781,3783, 3785, 3787, 3789, 3791, 3793, 3795, 3797, 3799, 3801, 3803, 3805,3807, 3809, 3811, 3813, 3815, 3817, 3819, 3821, 3823, 3825, 3827, 3829,3831, 3833, 3835, 3837, 3839, 3841, 3843, 3845, 3847, 3849, 3851, 3853,3855, 3857, 3859, 3861, 3863, 3865, 3867, 3869, 3871, 3873, 3875, 3877,3879, 3881, 3883, 3885, 3887, 3889, 3891, 3893, 3895, 3897, 3899, 3901,3903, 3905, 3907, 3909, 3911, 3913, 3915, 3917, 3919, 3921, 3923, 3925,3927, 3929, 3931, 3933, 3935, 3937, 3939, 3941, 3943, 3945, 3947, 3949,3951, 3953, 3955, 3957, 3959, 3961, 3963, 3965, 3967, 3969, 3971, 3973,3975, 3977, 3979, 3981, 3983, 3985, 3987, 3989, 3991, 3993, 3995, 3997,3999, 4001, 4003, 4005, 4007, 4009, 4011, 4013, 4015, 4017, 4019, 4021,4023, 4025, 4027, 4029, 4031, 4033, 4035, 4037, 4039, 4041, 4043, 4045,4047, 4049, 4051, 4053, 4055, 4057, 4059, 4061, 4063, 4065, 4067, 4069,4071, 4073, 4075, 4077, 4079, 4081, 4083, 4085, 4087, 4089, 4091, 4093,4095, 4097, 4099, 4101, 4103, 4105, 4107, 4109, 4111, 4113, 4115, 4117,4119, 4121, 4123, 4125, 4127, 4129, 4131, 4133, 4135, 4137, 4139, 4141,4143, 4145, 4147, 4149, 4151, 4153, 4155, 4157, 4159, 4161, 4163, 4165,4167, 4169, 4171, 4173, 4175, 4177, 4179, 4181, 4183, 4185, 4187, 4189,4191, 4193, 4195, 4197, 4199, 4201, 4203, 4205, 4207, 4209, 4211, 4213,4215, 4217, 4219, 4221, 4223, 4225, 4227, 4229, 4231, 4233, 4235, 4237,4239, 4241, 4243, 4245, 4247, 4249, 4251, 4253, 4255, 4257, 4259, 4261,4263, 4265, 4267, 4269, 4271, 4273, 4275, 4277, 4279, 4281, 4283, 4285,4287, 4289, 4291, 4293, 4295, 4297, 4299, 4301, 4303, 4305, 4307, 4309,4311, 4313, 4315, 4317, 4319, 4321, 4323, 4325, 4327, 4329, 4331, 4333,4335, 4337, 4339, 4341, 4343, 4345, 4347, 4349, 4351, 4353, 4355, 4357,4359, 4361, 4363, 4365, 4367, 4369, 4371, 4373, 4375, 4377, 4379, 4381,4383, 4385, 4387, 4389, 4391, 4393, 4395, 4397, 4399, 4401, 4403, 4405,4407, 4409, 4411, 4413, 4415, 4417, 4419, 4421, 4423, 4425, 4427, 4429,4431, 4433, 4435, 4437, 4439, 4441, 4443, 4445, 4447, 4449, 4451, 4453,4455, 4457, 4459, 4461, 4463, 4465, 4467, 4469, 4471, 4473, 4475, 4477,4479, 4481, 4483, 4485, 4487, 4489, 4491, 4493, 4495, 4497, 4499, 4501,4503, 4505, 4507, 4509, 4511, 4513, 4515, 4517, 4519, 4521, 4523, 4525,4527, 4529, 4531, 4533, 4535, 4537, 4539, 4541, 4543, 4545, 4547, 4549,4551, 4553, 4555, 4557, 4559, 4561, 4563, 4565, 4567, 4569, 4571, 4573,4575, 4577, 4579, 4581, 4583, 4585, 4587, 4589, 4591, 4593, 4595, 4597,4599, 4601, 4603, 4605, 4607, 4609, 4611, 4613, 4615, 4617, 4619, 4621,4623, 4625, 4627, 4629, 4631, 4633, 4635, 4637, 4639, 4641, 4643, 4645,4647, 4649, 4651, 4653, 4655, 4657, 4659, 4661, 4663, 4665, 4667, 4669,4671, 4673, 4675, 4677, 4679, 4681, 4683, 4685, 4687, 4689, 4691, 4693,4695, 4697, 4699, 4701, 4703, 4705, 4707, 4709, 4711, 4713, 4715, 4717,4719, 4721, 4723, 4725, 4727, 4729, 4731, 4733, 4735, 4737, 4739, 4741,4743, 4745, 4747, 4749, 4751, 4753, 4755, 4757, 4759, 4761, 4763, 4765,4767, 4769, 4771, 4773, 4775, 4777, 4779, 4781, 4783, 4785, 4787, 4789,4791, 4793, 4795, 4797, 4799, 4801, 4803, 4805, 4807, 4809, 4811, 4813,4815, 4817, 4819, 4821, 4823, 4825, 4827, 4829, 4831, 4833, 4835, 4837,4839, 4841, 4843, 4845, 4847, 4849, 4851, 4853, 4855, 4857, 4859, 4861,4863, 4865, 4867, 4869, 4871, 4873, 4875, 4877, 4879, 4881, 4883, 4885,4887, 4889, 4891, 4893, 4895, 4897, 4899, 4901, 4903, 4905, 4907, 4909,4911, 4913, 4915, 4917, 4919, 4921, 4923, 4925, 4927, 4929, 4931, 4933,4935, 4937, 4939, 4941, 4943, 4945, 4947, 4949, 4951, 4953, 4955, 4957,4959, 4961, 4963, 4965, 4967, 4969, 4971, 4973, 4975, 4977, 4979, 4981,4983, 4985, 4987, 4989, 4991, 4993, 4995, 4997, 4999, 5001, 5003, 5005,5007, 5009, 5011, 5013, 5015, 5017, 5019, 5021, 5023, 5025, 5027, 5029,5031, 5033, 5035, 5037, 5039, 5041, 5043, 5045, 5047, 5059, 5051, 5053,5055, 5057, 5059, 5061, 5063, 5065, 5067, 5069, 5071, 5073, 5075, 5077,5079, 5081, 5083, 5085, 5087, 5089, 5091, 5093, 5095, 5097, 5099, 5101,5103, 5105, 5107, 5109, 5111, 5113, 5115, 5117, 5119, 5121, 5123, 5125,5127, 5129, 5131, 5133, 5135, 5137, 5139, 5141, 5143, 5145, 5147, 5149,5151, 5153, 5155, 5157, 5159, 5161, 5163, 5165, 5167, 5169, 5171, 5173,5175, 5177, 5179, 5181, 5183, 5185, 5187, 5189, 5191, 5193, 5195, 5197,5199, 5201, 5203, 5205, 5207, 5209, 5211, 5213, 5215, 5217, 5219, 5221,5223, 5225, 5227, 5229, 5231, 5233, 5235, 5237, 5239, 5241, 5243, 5245,5247, 5249, 5251, 5253, 5255, 5257, 5259, 5261, 5263, 5265, 5267, 5269,5271, 5273, 5275, 5277, 5279, 5281, 5283, 5285, 5287, 5289, 5291, 5293,5295, 5297, 5299, 5301, 5303, 5305, 5307, 5309, 5311, 5313, 5315, 5317,5319, 5321, 5323, 5325, 5327, 5329, 5331, 5333, 5335, 5337, 5339, 5341,5343, 5345, 5347, 5349, 5351, 5353, 5355, 5357, 5359, 5361, 5363, 5365,5367, 5369, 5371, 5373, 5375, 5377, 5379, 5381, 5383, 5385, 5387, 5389,5391, 5393, 5395, 5397, 5399, 5401, 5403, 5405, 5407, 5409, 5411, 5413,5415, 5417, 5419, 5421, 5423, 5425, 5427, 5429, 5431, 5433, 5435, 5437,5439, 5441, 5443, 5445, 5447, 5449, 5451, 5453, 5455, 5457, 5459, 5461,5463, 5465, 5467, 5469, 5471, 5473, 5475, 5477, 5479, 5481, 5483, 5485,5487, 5489, 5491, 5493, 5495, 5497, 5499, 5501, 5503, 5505, 5507, 5509,5511, 5513, 5515, 5517, 5519, 5521, 5523, 5525, 5527, 5529, 5531, 5533,5535, 5537, 5539, 5541, 5543, 5545, 5547, 5549, 5551, 5553, 5555, 5557,5559, 5561, 5563, 5565, 5567, 5569, 5571, 5573, 5575, 5577, 5579, 5581,5583, 5585, 5587, 5589, 5591, 5593, 5595, 5597, 5599, 5601, 5603, 5605,5607, 5609, 5611, 5613, 5615, 5617, 5619, 5621, 5623, 5625, 5627, 5629,5631, 5633, 5635, 5637, 5639, 5641, 5643, 5645, 5647, 5649, 5651, 5653,5655, 5657, 5659, 5661, 5663, 5665, 5667, 5669, 5671, 5673, 5675, 5677,5679, 5681, 5683, 5685, 5687, 5689, 5691, 5693, 5695, 5697, 5699, 5701,5703, 5705, 5707, 5709, 5711, 5713, 5715, 5717, 5719, 5721, 5723, 5725,5727, 5729, 5731, 5733, 5735, 5737, 5739, 5741, 5743, 5745, 5747, 5749,5751, 5753, 5755, 5757, 5759, 5761, 5763, 5765, 5767, 5769, 5771, 5773,5775, 5777, 5779, 5781, 5783, 5785, 5787, 5789, 5791, 5793, 5795, 5797,5799, 5801, 5803, 5805, 5807, 5809, 5811, 5813, 5815, 5817, 5819, 5821,5823, 5825, 5827, 5829, 5831, 5833, 5835, 5837, 5839, 5841, 5843, 5845,5847, 5849, 5851, 5853, 5855, 5857, 5859, 5861, 5863, 5865, 5867, 5869,5871, 5873, 5875, 5877, 5879, 5881, 5883, 5885, 5887, 5889, 5891, 5893,5895, 5897, 5899, 5901, 5903, 5905, 5907, 5909, 5911, 5913, 5915, 5917,5919, 5921, 5923, 5925, 5927, 5929, 5931, 5933, 5935, 5937, 5939, 5941,5943, 5945, 5947, 5949, 5951, 5953, 5955, 5957, 5959, 5961, 5963, 5965,5967, 5969, 5971, 5973, 5975, 5977, 5979, 5981, 5983, 5985, 5987, 5989,5991, 5993, 5995, 5997, 5999, 6001, 6003, 6005, 6007, 6009, 6011, 6013,6015, 6017, 6019, 6021, 6023, 6025, 6027, 6029, 6031, 6033, 6035, 6037,6039, 6041, 6043, 6045, 6047, 6049, 6051, 6053, 6055, 6057, 6059, 6061,6063, 6065, 6067, 6069, 6071, 6073, 6075, 6077, 6079, 6081, 6083, 6085,6087, 6089, 6091, 6093, 6095, 6097, 6099, 6101, 6103, 6105, 6107, 6109,6111, 6113, 6115, 6117, 6119, 6121, 6123, 6125, 6127, 6129, 6131, 6133,6135, 6137, 6139, 6141, 6143, 6145, 6147, 6149, 6151, 6153, 6155, 6157,6159, 6161, 6163, 6165, 6167, 6169, 6171, 6173, 6175, 6177, 6179, 6181,6183, 6185, 6187, 6189, 6191, 6193, 6195, 6197, 6199, 6201, 6203, 6205,6207, 6209, 6211, 6213, 6215, 6217, 6219, 6221, 6223, 6225, 6227, 6229,6231, 6233, 6235, 6237, 6239, 6241, 6243, 6245, 6247, 6249, 6251, 6253,6255, 6257, 6259, 6261, 6263, 6265, 6267, 6269, 6271, 6273, 6275, 6277,6279, 6281, 6283, 6285, 6287, 6289, 6291, 6293, 6295, 6297, 6299, 6301,6303, 6305, 6307, 6309, 6311, 6313, 6315, 6317, 6319, 6321, 6323, 6325,6327, 6329, 6331, 6333, 6335, 6337, 6339, 6341, 6343, 6345, 6347, 6349,6351, 6353, 6355, 6357, 6359, 6361, 6363, 6365, 6367, 6369, 6371, 6373,6375, 6377, 6379, 6381, 6383, 6385, 6387, 6389, 6391, 6393, 6395, 6397,6399, 6401, 6403, 6405, 6407, 6409, 6411, 6413, 6415, 6417, 6419, 6421,6423, 6425, 6427, 6429, 6431, 6433, 6435, 6437, 6439, 6441, 6443, 6445,6447, 6449, 6451, 6453, 6455, 6457, 6459, 6461, 6463, 6465, 6467, 6469,6471, 6473, 6475, 6477, 6479, 6481, 6483, 6485, 6487, 6489, 6491, 6493,6495, 6497, 6499, 6501, 6503, 6505, 6507, 6509, 6511, 6513, 6515, 6517,6519, 6521, 6523, 6525, 6527, 6529, 6531, 6533, 6535, 6537, 6539, 6541,6543, 6545, 6547, 6549, 6551, 6553, 6555, 6557, 6559, 6561, 6563, 6565,6567, 6569, 6571, 6573, 6575, 6577, 6579, 6581, 6583, 6585, 6587, 6589,6591, 6593, 6595, 6597, 6599, 6601, 6603, 6605, 6607, 6609, 6611, 6613,6615, 6617, 6619, 6621, 6623, 6625, 6627, 6629, 6631, 6633, 6635, 6637,6639, 6641, 6643, 6645, 6647, 6649, 6651, 6653, 6655, 6657, 6659, 6661,6663, 6665, 6667, 6669, 6671, 6673, 6675, 6677, 6679, 6681, 6683, 6685,6687, 6689, 6691, 6693, 6695, 6697, 6699, 6701, 6703, 6705, 6707, 6709,6711, 6713, 6715, 6717, 6719, 6721, 6723, 6725, 6727, 6729, 6731, 6733,6735, 6737, 6739, 6741, 6743, 6745, 6747, 6749, 6751, 6753, 6755, 6757,6759, 6761, 6763, 6765, 6767, 6769, 6771, 6773, 6775, 6777, 6779, 6781,6783, 6785, 6787, 6789, 6791, 6793, 6795, 6797, 6799, 6801, 6803, 6805,6807, 6809, 6811, 6813, 6815, 6817, 6819, 6821, 6823, 6825, 6827, 6829,6831, 6833, 6835, 6837, 6839, 6841, 6843, 6845, 6847, 6849, 6851, 6853,6855, 6857, 6859, 6861, 6863, 6865, 6867, 6869, 6871, 6873, 6875, 6877,6879, 6881, 6883, 6885, 6887, 6889, 6891, 6893, 6895, 6897, 6899, 6901,6903, 6905, 6907, 6909, 6911, 6913, 6915, 6917, 6919, 6921, 6923, 6925,6927, 6929, 6931, 6933, 6935, 6937, 6939, 6941, 6943, 6945, 6947, 6949,6951, 6953, 6955, 6957, 6959, 6961, 6963, 6965, 6967, 6969, 6971, 6973,6975, 6977, 6979, 6981, 6983, 6985, 6987, 6989, 6991, 6993, 6995, 6997,6999, 7001, 7003, 7005, 7007, 7009, 7011, 7013, 7015, 7017, 7019, 7021,7023, 7025, 7027, 7029, 7031, 7033, 7035, 7037, 7039, 7041, 7043, 7045,7047, 7049, 7051, 7053, 7055, 7057, 7059, 7061, 7063, 7065, 7067, 7069,7071, 7073, 7075, 7077, 7079, 7081, 7083, 7085, 7087, 7089, 7091, 7093,7095, 7097, 7099, 7101, 7103, 7105, 7107, 7109, 7111, 7113, 7115, 7117,7119, 7121, 7123, 7125, 7127, 7129, 7131, 7133, 7135, 7137, 7139, 7141,7143, 7145, 7147, 7149, 7151, 7153, 7155, 7157, 7159, 7161, 7163, 7165,7167, 7169, 7171, 7173, 7175, 7177, 7179, 7181, 7183, 7185, 7187, 7189,7191, 7193, 7195, 7197, 7199, 7201, 7203, 7205, 7207, 7209, 7211, 7213,7215, 7217, 7219, 7221, 7223, 7225, 7227, 7229, 7231, 7233, 7235, 7237,7239, 7241, 7243, 7245, 7247, 7249, 7251, 7253, 7255, 7257, 7259, 7261,7263, 7265, 7267, 7269, 7271, 7273, 7275, 7277, 7279, 7281, 7283, 7285,7287, 7289, 7291, 7293, 7295, 7297, 7299, 7301, 7303, 7305, 7307, 7309,7311, 7313, 7315, 7317, 7319, 7321, 7323, 7325, 7327, 7329, 7331, 7333,7335, 7337, 7339, 7341, 7343, 7345, 7347, 7349, 7351, 7353, 7355, 7357,7359, 7361, 7363, 7365, 7367, 7369, 7371, 7373, 7375, 7377, 7379, 7381,7383, 7385, 7387, 7389, 7391, 7393, 7395, 7397, 7399, 7401, 7403, 7405,7407, 7409, 7411, 7413, 7415, 7417, 7419, 7421, 7423, 7425, 7427, 7429,7431, 7433, 7435, 7765, 7767, 7769, 7771, 7773, 7775, 7777, 7779, 7781,7783, 7785, 7787, 7789, 7791, 7793, 7795, 7797, 7799, 7811, 7813, 7815,7817, 7819, 7821, 7823, 7825, 7827, 7829, 7831, 7833, 7835, 7837, 7839,7841, 7843, 7845, 7847, 7849, 7851, 7853, 7855, 7857, 7859, 7861, 7863,7865, 7867, 7869, 7871, 7873, 7875, 7877, 7879, 7881, 7883, 7885, 7887,7889, 7891, 7893, 7895, 7897, 7899, 7901, 7903, 7905, 7907, 7909, 7911,7913, 7915, 7917, 7919, 7921, 7923, 7925, 7927, 7929, 7931, 7933, 7935,7937, 7939, 7941, 7943, 7945, 7947, 7949, 7951, 7953, 7955, 7957, 7959,7961, 7963, 7965, 7967, 7969, 7971, 7973, 7975, 7977, 7979, 7981, 7983,7985, 7987, 7989, 7991, 7993, 7995, 7997, 7999, 8001, 8003, 8005, 8007,8009, 8011, 8013, 8015, 8017, 8019, 8021, 8023, 8025, 8027, 8029, 8031,8033, 8035, 8037, 8039, 8041, 8043, 8045, 8047, 8049, 8051, 8053, 8055,8057, 8059, 8061, 8063, 8065, 8067, 8069, 8071, 8073, 8075, 8077, 8079,8081, 8083, 8085, 8087, 8089, 8091, 8093, 8095, 8097, 8099, 8101, 8103,8105, 8107, 8109, 8111, 8113, 8115, 8117, 8119, 8121, 8123, 8125, 8127,8129, 8131, 8133, 8135, 8137, 8139, 8141, 8143, 8145, 8147, 8149, 8151,8153, 8155, 8157, 8159, 8161, 8163, 8165, 8167, 8169, 8171, 8173, 8175,8177, 8179, 8181, 8183, 8185, 8187, 8189, 8191, 8193, 8195, 8197, 8199,8201, 8203, 8205, 8207, 8209, 8211, 8213, 8215, 8217, 8219, 8221, 8223,8225, 8227, 8229, 8231, 8233, 8235, 8237, 8239, 8241, 8243, 8245, 8247,8249, 8251, 8253, 8255, 8257, 8259, 8261, 8263, 8265, 8267, 8269, 8271,8273, 8275, 8277, 8279, 8281, 8283, 8285, 8287, 8289, 8291, 8293, 8295,8297, 8299, 8301, 8303, 8305, 8307, 8309, 8311, 8313, 8315, 8317, 8319,8321, 8323, 8325, 8327, 8329, 8331, 8333, 8335, 8337, 8339, 8341, 8343,8345, 8347, 8349, 8351, 8353, 8355, 8357, 8359, 8361, 8363, 8365, 8367,8481, 8483, 8485, 8487, 8489, 8491, 8493, 8495, 8497, 8489, 8501, 8503,8505, 8507, 8509, 8511, 8513, 8515, 8517, 8519, 8521, 8523, 8525, 8527,8529, 8531, 8533 8535, 8537, 8539, 8541, 8543, 8545, 8547, 8549, 8551,8553, 8555, 8557, 8559, 8561, 8563, 8565, 8567, 8569, 8571, 8573, 8575,8577, 8579, 8581, 8583, 8585, 8587, 8589, 8591, 8593, 8595, 8597, 8599,8601, 8603, 8605, 8607, 8609, 8611, 8613, 8615, 8617, 8619, 8621, 8623,8625, 8627, 8629, 8631, 8633, 8635, 8637, 8639, 8641, 8643, 8645, 8647,8649, 8651, 8653, 8655, 8657, 8659, 8661, 8663, 8665, 8667, 8669, 8671,8673, 8675, 8677, 8679, 8681, 8683, 8685, 8687, 8689, 8691, 8693, 8695,8697, 8699, 8701, 8703, 8705, 8707, 8709, 8711, 8713, 8715, 8717, 8719,8721, 8723, 8725, 8727, 8729, 8731, 8733, 8735, 8737, 8739, 8741, 8743,8745, 8747, 8749, 8751, 8753, 8755, 8757, 8759, 8761, 8763, 8765, 8767,8769, 8771, 8773, 8775, 8777, 8779, 8781, 8783, 8785, 8787, 8789, 8791,8793, 8795, 9107, 9109, 9111, 9113, 9115, 9117, 9119, 9121, 9123, 9125,9127, 9129, 9131, 9133, 9135, 9137, 9139, 9141, 9143, 9145, 9147, 9149,9151, 9153, 9155, 9157, 9159, 9161, 9163, 9165, 9167, 9169, 9171, 9173,9175, 9177, 9179, 9181, 9183, 9185, 9187, 9189, 9191, 9193, 9195, 9197,9199, 9201, 9203, 9205, 9207, 9209, 9211, 9213, 9215, 9217, 9219, 9221,9223, 9225, 9227, 9229, 9231, 9233, 9235, 9237, and/or 9239.

In some additional embodiments, the engineered polynucleotide encodingat least one engineered sucrose synthase provided herein comprises atleast 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to SEQ IDNO:1079, 1081, 1083, 1085, 1087, 1089, 1091, 1093, 1095, 1097, 1099,1101, 1103, 1105, 1107, 1109, 1111, 1113, 1115, 1117, 1119, 1121, 1123,1125, 1127, 1129, 1131, 1133, 1135, 1137, 1139, 1141, 1143, 1145, 1147,1149, 1151, 1153, 1155, 1157, 1159, 1161, 1163, 1165, 1167, 1169, 1171,1173, 1175, 1177, 1179, 1181, 1183, 1185, 1187, 1189, 1191, 1193, 1195,1197, 1199, 1201, 1203, 1205, 1207, 1209, 1211, 1213, 1215, 1217, 1219,1221, 1223, 1225, 1227, 1229, 1231, 1233, 1235, 1237, 1239, 1241, 1243,1245, 1247, 1249, 1251, 1253, 1255, 1257, 1259, 1261, 1263, 1265, 1267,1269, 1271, 1273, 1275, 1277, 1279, 1281, 1283, 1285, 1287, 1295, 1297,1299, 1301, 1303, 1305, 1307, 1309, 1311, 1313, 1315, 1317, 1319, 1321,1323, 1325, 1327, 1329, 1331, 1333, 1335, 1337, 1339, 1341, 1343, 1345,1347, 1349, 1351, 1353, 1355, 1357, 1359, 1361, 1363, 1365, 1367, 1369,1371, 1373, 1375, 1377, 1379, 1381, 1383, 1385, 1387, 1389, 1391, 1393,1395, 1397, 1399, 1401, 1403, 1405, 1407, 1409, 1411, 1413, 1415, 1417,1419, 1421, 1423, 1425, 1427, 1429, 1431, 1433, 1435, 1437, 1439, 1441,1443, 1445, 1447, 1449, 1451, 1453, 1455, 1457, 1459, 1461, 1463, 1465,1467, 1469, 1471, 1473, 1475, 1477, 1479, 1481, 1483, 1485, 1487, 1489,1491, 1493, 1495, 1497, 1499, 1501, 1503, 1505, 1507, 1509, 1511, 1513,1515, 1517, 1519, 1521, 1523, 1525, 1527, 1529, 1531, 1533, 1535, 1537,1539, 1541, 1543, 1545, 1547, 1549, 1551, 1553, 1555, 1557, 1559, 1561,1563, 1565, 1567, 1569, 1571, 1573, 1575, 1577, 1579, 1581, 1583, 1585,1587, 1589, 1591, 1593, 1595, 1597, 1599, 1601, 1603, 1605, 1607, 1609,1611, 1613, 1615, 1617, 1619, 1621, 1623, 1625, 1627, 1629, 1631, 1633,1635, 1637, 1639, 1641, 1643, 1645, 1647, 1649, 1651, 1653, 1655, 1657,1659, 1661, 1663, 1665, 1667, 1669, 1671, 1673, 1675, 1677, 1679, 1681,1683, 1685, 1687, 1689, 1691, 1693, 1695, 1697, 1699, 1701, 1703, 1705,1707, 1709, 1711, 1713, 1715, 1717, 1719, 1721, 1723, 1725, 1727, 1729,1731, 1733, 1735, 1737, 1739, 1741, 1743, 1745, 1747, 1749, 1751, 1753,1755, 1757, 1759, 1761, 1763, 1765, 1767, 1769, 1771, 1773, 1775, 1777,1779, 1781, 1783, 1785, 1787, 1789, 1791, 1793, 1795, 1797, 1799, 1801,1803, 1805, 1807, 1809, 1811, 1813, 1815, 1817, 1819, 1821, 1823, 1825,1827, 1829, 1831, 1833, 1835, 1837, 1839, 1841, 1843, 1845, 1847, 1849,1851, 1853, 1855, 1857, 1859, 1861, 1863, 1865, 1867, 1869, 1871, 1873,1875, 1877, 1879, 1881, 1883, 1885, 1887, 1889, 1891, 1893, 1895, 1897,1899, 1901, 1903, 1905, 1907, 1909, 1911, 1913, 1915, 1917, 1919, 1921,1923, 1925, 1927, 1929, 1931, 1933, 1935, 1937, 1939, 1941, 1943, 1945,1947, 1949, 1951, 1953, 1955, 1957, 1959, 1961, 1963, 1965, 1967, 1969,1971, 1973, 1975, 1977, 1979, 1981, 1983, 1985, 1987, 1989, 1991, 1993,1995, 1997, 1999, 2001, 2003, 2005, 2007, 2009, 2011, 2013, 2015, 2017,2019, 2021, 2023, 2025, 2027, 2029, 2031, 2033, 2035, 2037, 2039, 2041,2043, 2045, 2047, 2049, 2051, 2053, 2055, 2057, 2059, 2061, 2063, 2065,2067, 2069, 2071, 2073, 2075, 2077, 2079, 2081, 2083, 2085, 2087, 2089,2091, 2093, 2095, 2097, 2099, 2101, 2103, 2105, 2107, 2109, 2111, 2113,2115, 2117, 2119, 2121, 2123, 2125, 2127, 2129, 2131, 2133, 2135, 2137,2139, 2141, 2143, 2145, 2147, 2149, 2151, 2153, 2155, 2157, 2159, 2161,2163, 2165, 2167, 2169, 2171, 2173, 2175, 2177, 2179, 2181, 2183, 2185,2187, 2189, 2191, 2193, 2195, 2197, 2199, 2201, 2203, 2205, 2207, 2209,2211, 2213, 2215, 2217, 2219, 2221, 2223, 2225, 2227, 2229, 2231, 2233,2235, 2237, 2239, 2241, 2243, 2245, 2247, 2249, 2251, 2253, 2255, 2257,2259, 2261, 2263, 2265, 2267, 2269, 2271, 2273, 2275, 2277, 2279, 2281,2283, 2285, 2287, 2289, 2291, 2293, 2295, 2297, 2299, 2301, 2303, 2305,2307, 2309, 2311, 2313, 2315, 2317, 2319, 2321, 2323, 2325, 2327, 2329,2331, 2333, 2335, 2337, 2339, 2341, 2343, 2345, 2347, 2349, 2351, 2353,2355, 2357, 2359, 2361, 2363, 2365, 2367, 2369, 2371, 2373, 2375, 2377,2379, 2381, 2383, 2385, 2387, 2389, 2391, 2393, 2395, 2397, 2399, 2401,2403, 2405, 2407, 2409, 2411, 2413, 2415, 2417, 2419, 2421, 2423, 2425,2427, 2429, 2431, 2433, 2435, 2437, 2439, 2441, 2443, 2445, 2447, 2449,2451, 2453, 2455, 2457, 2459, 2461, 2463, 2465, 2467, 2469, 2471, 2473,2475, 2477, 2479, 2481, 2483, 2485, 2487, 2489, 2491, 2493, 2495, 2497,2499, 2501, 2503, 2505, 2507, 2509, 2511, 2513, 2515, 2517, 2519, 2521,2523, 2525, 2527, 2529, 2531, 2533, 2535, 2537, 2539, 2541, 2543, 2545,2547, 2549, 2551, 2553, 2555, 2557, 2559, 2561, 2563, 2565, 2567, 2569,2571, 2573, 2575, 2577, 2579, 2581, 2583, 2585, 2587, 2589, 2591, 2593,7437, 7439, 7441, 7443, 7445, 7447, 7449, 7451, 7453, 7455, 7457, 7459,7461, 7463, 7465, 7467, 7469, 7471, 7473, 7475, 7477, 7479, 7481, 7483,7485, 7487, 7489, 7491, 7493, 7495, 7497, 7499, 7501, 7503, 7505, 7507,7509, 7511, 7513, 7515, 7517, 7519, 7521, 7523, 7525, 7527, 7529, 7531,7533, 7535, 7537, 7539, 7541, 7543, 7545, 7547, 7549, 7551, 7553, 7555,7557, 7559, 7561, 7563, 7565, 7567, 7569, 7571, 7573, 7575, 7577, 7579,7581, 7583, 7585, 7587, 7589, 7591, 7593, 7595, 7597, 7599, 7601, 7603,7605, 7607, 7609, 7611, 7613, 7615, 7617, 7619, 7621, 7623, 7625, 7627,7629, 7631, 7633, 7635, 7637, 7639, 7641, 7643, 7645, 7647, 7649, 7651,7653, 7655, 7657, 7659, 7661, 7663, 7665, 7667, 7669, 7671, 7673, 7675,7677, 7679, 7681, 7683, 7685, 7687, 7689, 7691, 7693, 7695, 7697, 7699,7701, 7703, 7705, 7707, 7709, 7711, 7713, 7715, 7717, 7719, 7721, 7723,7725, 7727, 7729, 7731, 7733, 7735, 7737, 7739, 7741, 7743, 7745, 7747,7749, 7751, 7753, 7755, 7757, 7759, 7761, 7763, 8369, 8371, 8373, 8375,8377, 8379, 8381, 8383, 8385, 8387, 8389, 8391, 8393, 8395, 8397, 8399,8401, 8403, 8405, 8407, 8409, 8411, 8413, 8415, 8417, 8419, 8421, 8423,8425, 8427, 8429, 8431, 8433, 8435, 8437, 8439, 8441, 8443, 8445, 8447,8449, 8451, 8453, 8455, 8457, 8459, 8461, 8463, 8465, 8467, 8469, 8471,8473, 8475, 8477, 8479, 8797, 8799, 8801, 8803, 8805, 8807, 8809, 8811,8813, 8815, 8817, 8819, 8821, 8823, 8825, 8827, 8829, 8831, 8833, 8835,8837, 8839, 8841, 8843, 8845, 8847, 8849, 8851, 8853, 8855, 8857, 8859,8861, 8863, 8865, 8867, 8869, 8871, 8873, 8875, 8877, 8879, 8881, 8883,8885, 8887, 8889, 8891, 8893, 8895, 8897, 8899, 8901, 8903, 8905, 8907,8909, 8911, 8913, 8915, 8917, 8919, 8921, 8923, 8925, 8927, 8929, 8931,8933, 8935, 8937, 8939, 8941, 8943, 8945, 8947, 8949, 8951, 8953, 8955,8957, 8959, 8961, 8963, 8965, 8967, 8969, 8971, 8973, 8975, 8977, 8979,8981, 8983, 8985, 8987, 8989, 8991, 8993, 8995, 8997, 8999, 9001, 9003,9005, 9007, 9009, 9011, 9013, 9015, 9017, 9019, 9021, 9023, 9025, 9027,9029, 9031, 9033, 9035, 9037, 9039, 9041, 9043, 9045, 9047, 9049, 9051,9053, 9055, 9057, 9059, 9061, 9063, 9065, 9067, 9069, 9071, 9073, 9075,9077, 9079, 9081, 9083, 9085, 9087, 9089, 9091, 9093, 9095, 9097, 9099,9101, 9103, and/or 9105.

In some embodiments, the polynucleotides are capable of hybridizingunder highly stringent conditions to a reference polynucleotide sequenceselected from any polynucleotide sequence provided herein, or acomplement thereof, or a polynucleotide sequence encoding any of thevariant enzyme polypeptides provided herein. In some embodiments, thepolynucleotide capable of hybridizing under highly stringent conditionsencodes a enzyme polypeptide comprising an amino acid sequence that hasone or more residue differences as compared to a reference sequence.

In some embodiments, an isolated polynucleotide encoding any of theengineered enzyme polypeptides herein is manipulated in a variety ofways to facilitate expression of the enzyme polypeptide. In someembodiments, the polynucleotides encoding the enzyme polypeptidescomprise expression vectors where one or more control sequences ispresent to regulate the expression of the enzyme polynucleotides and/orpolypeptides. Manipulation of the isolated polynucleotide prior to itsinsertion into a vector may be desirable or necessary depending on theexpression vector utilized.

Techniques for modifying polynucleotides and nucleic acid sequencesutilizing recombinant DNA methods are well known in the art. In someembodiments, the control sequences include among others, promoters,leader sequences, polyadenylation sequences, propeptide sequences,signal peptide sequences, and transcription terminators. In someembodiments, suitable promoters are selected based on the host cellsselection. For bacterial host cells, suitable promoters for directingtranscription of the nucleic acid constructs of the present disclosure,include, but are not limited to promoters obtained from the E. coli lacoperon, Streptomyces coelicolor agarase gene (dagA), Bacillus subtilislevansucrase gene (sacB), Bacillus licheniformis alpha-amylase gene(amyL), Bacillus stearothermophilus maltogenic amylase gene (amyM),Bacillus amyloliquefaciens alpha-amylase gene (amyQ), Bacilluslicheniformis penicillinase gene (penP), Bacillus subtilis xylA and xylBgenes, and prokaryotic beta-lactamase gene (See e.g., Villa-Kamaroff etal., Proc. Natl Acad. Sci. USA 75: 3727-3731 [1978]), as well as the tacpromoter (See e.g., DeBoer et al., Proc. Natl Acad. Sci. USA 80: 21-25[1983]). Exemplary promoters for filamentous fungal host cells, include,but are not limited to promoters obtained from the genes for Aspergillusoryzae TAKA amylase, Rhizomucor miehei aspartic proteinase, Aspergillusniger neutral alpha-amylase, Aspergillus niger acid stablealpha-amylase, Aspergillus niger or Aspergillus awamori glucoamylase(glaA), Rhizomucor miehei lipase, Aspergillus oryzae alkaline protease,Aspergillus oryzae triose phosphate isomerase, Aspergillus nidulansacetamidase, and Fusarium oxysporum trypsin-like protease (See e.g., WO96/00787), as well as the NA2-tpi promoter (a hybrid of the promotersfrom the genes for Aspergillus niger neutral alpha-amylase andAspergillus oryzae triose phosphate isomerase), and mutant, truncated,and hybrid promoters thereof. Exemplary yeast cell promoters can be fromthe genes can be from the genes for Saccharomyces cerevisiae enolase(ENO-1), Saccharomyces cerevisiae galactokinase (GAL1), Saccharomycescerevisiae alcohol dehydrogenase/glyceraldehyde-3-phosphatedehydrogenase (ADH2/GAP), and Saccharomyces cerevisiae3-phosphoglycerate kinase. Other useful promoters for yeast host cellsare known in the art (See e.g., Romanos et al., Yeast 8:423-488 [1992]).

In some embodiments, the control sequence is also a suitabletranscription terminator sequence (i.e., a sequence recognized by a hostcell to terminate transcription). In some embodiments, the terminatorsequence is operably linked to the 3′ terminus of the nucleic acidsequence encoding the enzyme polypeptide. Any suitable terminator whichis functional in the host cell of choice finds use in the presentinvention. Exemplary transcription terminators for filamentous fungalhost cells can be obtained from the genes for Aspergillus oryzae TAKAamylase, Aspergillus niger glucoamylase, Aspergillus nidulansanthranilate synthase, Aspergillus niger alpha-glucosidase, and Fusariumoxysporum trypsin-like protease. Exemplary terminators for yeast hostcells can be obtained from the genes for Saccharomyces cerevisiaeenolase, Saccharomyces cerevisiae cytochrome C (CYC1), and Saccharomycescerevisiae glyceraldehyde-3-phosphate dehydrogenase. Other usefulterminators for yeast host cells are known in the art (See e.g., Romanoset al., supra).

In some embodiments, the control sequence is also a suitable leadersequence (i.e., a non-translated region of an mRNA that is important fortranslation by the host cell). In some embodiments, the leader sequenceis operably linked to the 5′ terminus of the nucleic acid sequenceencoding the enzyme polypeptide. Any suitable leader sequence that isfunctional in the host cell of choice find use in the present invention.Exemplary leaders for filamentous fungal host cells are obtained fromthe genes for Aspergillus oryzae TAKA amylase, and Aspergillus nidulanstriose phosphate isomerase. Suitable leaders for yeast host cells areobtained from the genes for Saccharomyces cerevisiae enolase (ENO-1),Saccharomyces cerevisiae 3-phosphoglycerate kinase, Saccharomycescerevisiae alpha-factor, and Saccharomyces cerevisiae alcoholdehydrogenase/glyceraldehyde-3-phosphate dehydrogenase (ADH2/GAP).

In some embodiments, the control sequence is also a polyadenylationsequence (i.e., a sequence operably linked to the 3′ terminus of thenucleic acid sequence and which, when transcribed, is recognized by thehost cell as a signal to add polyadenosine residues to transcribedmRNA). Any suitable polyadenylation sequence which is functional in thehost cell of choice finds use in the present invention. Exemplarypolyadenylation sequences for filamentous fungal host cells include, butare not limited to the genes for Aspergillus oryzae TAKA amylase,Aspergillus niger glucoamylase, Aspergillus nidulans anthranilatesynthase, Fusarium oxysporum trypsin-like protease, and Aspergillusniger alpha-glucosidase. Useful polyadenylation sequences for yeast hostcells are known (See e.g., Guo and Sherman, Mol. Cell. Bio.,15:5983-5990 [1995]).

In some embodiments, the control sequence is also a signal peptide(i.e., a coding region that codes for an amino acid sequence linked tothe amino terminus of a polypeptide and directs the encoded polypeptideinto the cell's secretory pathway). In some embodiments, the 5′ end ofthe coding sequence of the nucleic acid sequence inherently contains asignal peptide coding region naturally linked in translation readingframe with the segment of the coding region that encodes the secretedpolypeptide. Alternatively, in some embodiments, the 5′ end of thecoding sequence contains a signal peptide coding region that is foreignto the coding sequence. Any suitable signal peptide coding region whichdirects the expressed polypeptide into the secretory pathway of a hostcell of choice finds use for expression of the engineeredpolypeptide(s). Effective signal peptide coding regions for bacterialhost cells are the signal peptide coding regions include, but are notlimited to those obtained from the genes for Bacillus NCB 11837maltogenic amylase, Bacillus stearothermophilus alpha-amylase, Bacilluslicheniformis subtilisin, Bacillus licheniformis beta-lactamase,Bacillus stearothermophilus neutral proteases (nprT, nprS, nprM), andBacillus subtilis prsA. Further signal peptides are known in the art(See e.g., Simonen and Palva, Microbiol. Rev., 57:109-137 [1993]). Insome embodiments, effective signal peptide coding regions forfilamentous fungal host cells include, but are not limited to the signalpeptide coding regions obtained from the genes for Aspergillus oryzaeTAKA amylase, Aspergillus niger neutral amylase, Aspergillus nigerglucoamylase, Rhizomucor miehei aspartic proteinase, Humicola insolenscellulase, and Humicola lanuginosa lipase. Useful signal peptides foryeast host cells include, but are not limited to those from the genesfor Saccharomyces cerevisiae alpha-factor and Saccharomyces cerevisiaeinvertase.

In some embodiments, the control sequence is also a propeptide codingregion that codes for an amino acid sequence positioned at the aminoterminus of a polypeptide. The resultant polypeptide is referred to as a“proenzyme,” “propolypeptide,” or “zymogen.” A propolypeptide can beconverted to a mature active polypeptide by catalytic or autocatalyticcleavage of the propeptide from the propolypeptide. The propeptidecoding region may be obtained from any suitable source, including, butnot limited to the genes for Bacillus subtilis alkaline protease (aprE),Bacillus subtilis neutral protease (nprT), Saccharomyces cerevisiaealpha-factor, Rhizomucor miehei aspartic proteinase, and Myceliophthorathermophila lactase (See e.g., WO 95/33836). Where both signal peptideand propeptide regions are present at the amino terminus of apolypeptide, the propeptide region is positioned next to the aminoterminus of a polypeptide and the signal peptide region is positionednext to the amino terminus of the propeptide region.

In some embodiments, regulatory sequences are also utilized. Thesesequences facilitate the regulation of the expression of the polypeptiderelative to the growth of the host cell. Examples of regulatory systemsare those that cause the expression of the gene to be turned on or offin response to a chemical or physical stimulus, including the presenceof a regulatory compound. In prokaryotic host cells, suitable regulatorysequences include, but are not limited to the lac, tac, and trp operatorsystems. In yeast host cells, suitable regulatory systems include, butare not limited to the ADH2 system or GAL1 system. In filamentous fungi,suitable regulatory sequences include, but are not limited to the TAKAalpha-amylase promoter, Aspergillus niger glucoamylase promoter, andAspergillus oryzae glucoamylase promoter.

In another aspect, the present invention is directed to a recombinantexpression vector comprising a polynucleotide encoding an engineeredenzyme polypeptide, and one or more expression regulating regions suchas a promoter and a terminator, a replication origin, etc., depending onthe type of hosts into which they are to be introduced. In someembodiments, the various nucleic acid and control sequences describedherein are joined together to produce recombinant expression vectorswhich include one or more convenient restriction sites to allow forinsertion or substitution of the nucleic acid sequence encoding theenzyme polypeptide at such sites. Alternatively, in some embodiments,the nucleic acid sequence of the present invention is expressed byinserting the nucleic acid sequence or a nucleic acid constructcomprising the sequence into an appropriate vector for expression. Insome embodiments involving the creation of the expression vector, thecoding sequence is located in the vector so that the coding sequence isoperably linked with the appropriate control sequences for expression.

The recombinant expression vector may be any suitable vector (e.g., aplasmid or virus), that can be conveniently subjected to recombinant DNAprocedures and bring about the expression of the enzyme polynucleotidesequence. The choice of the vector typically depends on thecompatibility of the vector with the host cell into which the vector isto be introduced. The vectors may be linear or closed circular plasmids.

In some embodiments, the expression vector is an autonomouslyreplicating vector (i.e., a vector that exists as an extra-chromosomalentity, the replication of which is independent of chromosomalreplication, such as a plasmid, an extra-chromosomal element, aminichromosome, or an artificial chromosome). The vector may contain anymeans for assuring self-replication. In some alternative embodiments,the vector is one in which, when introduced into the host cell, it isintegrated into the genome and replicated together with thechromosome(s) into which it has been integrated.

Furthermore, in some embodiments, a single vector or plasmid, or two ormore vectors or plasmids which together contain the total DNA to beintroduced into the genome of the host cell, and/or a transposon isutilized.

In some embodiments, the expression vector contains one or moreselectable markers, which permit easy selection of transformed cells. A“selectable marker” is a gene, the product of which provides for biocideor viral resistance, resistance to heavy metals, prototrophy toauxotrophs, and the like. Examples of bacterial selectable markersinclude, but are not limited to the dal genes from Bacillus subtilis orBacillus licheniformis, or markers, which confer antibiotic resistancesuch as ampicillin, kanamycin, chloramphenicol or tetracyclineresistance. Suitable markers for yeast host cells include, but are notlimited to ADE2, HIS3, LEU2, LYS2, MET3, TRP1, and URA3. Selectablemarkers for use in filamentous fungal host cells include, but are notlimited to, amdS (acetamidase; e.g., from A. nidulans or A. orzyae),argB (ornithine carbamoyltransferases), bar (phosphinothricinacetyltransferase; e.g., from S. hygroscopicus), hph (hygromycinphosphotransferase), niaD (nitrate reductase), pyrG(orotidine-5′-phosphate decarboxylase; e.g., from A. nidulans or A.orzyae), sC (sulfate adenyltransferase), and trpC (anthranilatesynthase), as well as equivalents thereof. In another aspect, thepresent invention provides a host cell comprising at least onepolynucleotide encoding at least one engineered enzyme polypeptide ofthe present invention, the polynucleotide(s) being operatively linked toone or more control sequences for expression of the engineered enzymeenzyme(s) in the host cell. Host cells suitable for use in expressingthe polypeptides encoded by the expression vectors of the presentinvention are well known in the art and include but are not limited to,bacterial cells, such as E. coli, Vibrio fluvialis, Streptomyces andSalmonella typhimurium cells; fungal cells, such as yeast cells (e.g.,Saccharomyces cerevisiae or Pichia pastoris (ATCC Accession No.201178)); insect cells such as Drosophila S2 and Spodoptera Sf9 cells;animal cells such as CHO, COS, BHK, 293, and Bowes melanoma cells; andplant cells. Exemplary host cells also include various Escherichia colistrains (e.g., W3110 (AfhuA) and BL21).

Accordingly, in another aspect, the present invention provides methodsof producing the engineered enzyme polypeptides, where the methodscomprise culturing a host cell capable of expressing a polynucleotideencoding the engineered enzyme polypeptide under conditions suitable forexpression of the polypeptide. In some embodiments, the methods furthercomprise the steps of isolating and/or purifying the enzymepolypeptides, as described herein.

Appropriate culture media and growth conditions for host cells are wellknown in the art. It is contemplated that any suitable method forintroducing polynucleotides for expression of the enzyme polypeptidesinto cells will find use in the present invention. Suitable techniquesinclude, but are not limited to electroporation, biolistic particlebombardment, liposome mediated transfection, calcium chloridetransfection, and protoplast fusion.

Various features and embodiments of the present invention areillustrated in the following representative examples, which are intendedto be illustrative, and not limiting.

Experimental

The following Examples, including experiments and results achieved, areprovided for illustrative purposes only and are not to be construed aslimiting the present invention. Indeed, there are various suitablesources for many of the reagents and equipment described below. It isnot intended that the present invention be limited to any particularsource for any reagent or equipment item.

In the experimental disclosure below, the following abbreviations apply:M (molar); mM (millimolar), uM and M (micromolar); nM (nanomolar); mol(moles); gm and g (gram); mg (milligrams); ug and μg (micrograms); L and1 (liter); ml and mL (milliliter); cm (centimeters); mm (millimeters);um and μm (micrometers); sec. (seconds); min(s) (minute(s)); h(s) andhr(s) (hour(s)); U (units); MW (molecular weight); rpm (rotations perminute); psi and PSI (pounds per square inch); ° C. (degreesCentigrade); RT and rt (room temperature); CV (coefficient ofvariability); CAM and cam (chloramphenicol); PMBS (polymyxin B sulfate);IPTG (isopropyl β-D-1-thiogalactopyranoside); LB (Luria broth); TB(terrific broth); SFP (shake flask powder); CDS (coding sequence); DNA(deoxyribonucleic acid); RNA (ribonucleic acid); nt (nucleotide;polynucleotide); aa (amino acid; polypeptide); E. coli W3110 (commonlyused laboratory E. coli strain, available from the Coli Genetic StockCenter [CGSC], New Haven, Conn.); AcSus (Acidithiobacillus caldussucrose synthase); SUS, SuS, and SuSy (sucrose synthase, also known assucrose synthetase); NDP (nucleoside diphosphate); adenosine diphosphate(ADP); cytidine diphosphate (CDP); guanosine diphosphate (GDP);thymidine diphosphate (TDP); uridine diphosphate (UDP); inosinediphosphate (IDP); GT (glycosyltransferase); UGT (UDP-glucose-dependentglycosyltransferase); NGT (NDP-nucleoside diphosphate-dependentglycosyltransferase); AGT (ADP-glucose-dependent glycosyltransferase);CGT (CDP-glucose-dependent glycosyltransferase); GGT(GDP-glucose-dependent glycosyltransferase); TGT (TDP-glucose-dependentglycosyltransferase); IGT (IDP-glucose-dependent glycosyltransferase);UGT (UDP-glucose-dependent glycosyltransferase); reb (rebaudioside);rebA (rebaudioside A); rebD (rebaudioside D); rebI (rebaudioside I);rebM (rebaudioside M); “Reb A 60” is a ˜1:2 mixture of stevioside andrebaudioside A respectively; HTP (high throughput); HPLC (high pressureliquid chromatography); HPLC-UV (HPLC-Ultraviolet Visible Detector); 1HNMR (proton nuclear magnetic resonance spectroscopy); HSQC NMR(heteronuclear single quantum coherence spectroscopy NMR); COSY NMR(homonuclear correlation spectroscopy NMR); Acorn (Acorn NMR, Livermore,Calif.); FIOPC (fold improvements over positive control); Sigma andSigma-Aldrich (Sigma-Aldrich, St. Louis, Mo.; Difco (Difco Laboratories,BD Diagnostic Systems, Detroit, Mich.); Microfluidics (Microfluidics,Westwood, Mass.); ChromaDex (ChromaDex, Inc., Irvine, Calif.); LifeTechnologies (Life Technologies, a part of Fisher Scientific, Waltham,Mass.); Amresco (Amresco, LLC, Solon, Ohio); Carbosynth (Carbosynth,Ltd., Berkshire, UK); Varian (Varian Medical Systems, Palo Alto,Calif.); Agilent (Agilent Technologies, Inc., Santa Clara, Calif.); andThermotron (Thermotron, Inc., Holland, Mich.).

Example 1 Synthesis, Optimization, and Assaying of UGT Enzymes withGlucosylation Activity

In this Example, methods used in the synthesis, optimization andassaying of UGT enzymes with glucosylation activity are described.

Gene Synthesis and Optimization:

The polynucleotide sequence (SEQ ID NO: 1) encoding the wild-type Steviarebaudiana polypeptide (SEQ ID NO: 2) reported to glucosylatesteviolbioside to rebaudioside B and glucosylate stevioside torebaudioside A (See e.g., Richman et al., Plant J., 41:56-67 [2005]),was codon-optimized and synthesized as the gene of SEQ ID NO: 3. Thissynthetic gene (SEQ ID NO: 3) was cloned into a pCK110900 vector system(See e.g., US Pat. Appln. Pubn. No. 2006/0195947, which is herebyincorporated by reference herein) and subsequently expressed in E. coliW3110 (AfhuA). The E. coli strain W3110 expressed the UGT enzymes underthe control of the lac promoter.

Production of Shake Flask Powders (SFP):

A shake-flask procedure was used to generate the glycosyltransferasepolypeptide shake flask powders (SFP) for characterization assays usedin the biocatalytic processes described herein. Shake flask powder (SFP)preparation of enzymes provides a more purified preparation (e.g., upto >30% of total protein) of the enzyme as compared to the cell lysateused in HTP assays and also allows for the use of more concentratedenzyme solutions. A single colony of E. coli containing a plasmidencoding an engineered polypeptide of interest was inoculated into 5 mLLuria Bertani broth containing 30 μg/ml chloramphenicol and 1% glucose.Cells were grown overnight (at least 16 hours) in an incubator at 30° C.with shaking at 250 rpm. The culture was diluted into 250 mL TerrificBroth (12 g/L bacto-tryptone, 24 g/L yeast extract, 4 mL/L glycerol, 65mM potassium phosphate, pH 7.0, 1 mM MgS4) containing 30 μg/ml CAM, in a1 L flask to an optical density of 600 nm (OD600) of 0.2 and allowed togrow at 30° C.

Expression of the glycosyltransferase gene was induced by addition ofIPTG to a final concentration of 1 mM when the OD600 of the culture was0.6 to 0.8. Incubation was then continued overnight (at least 16 hours).Cells were harvested by centrifugation (5000 rpm, 15 min, 4° C.) and thesupernatant discarded. The cell pellet was resuspended in two volumes of25 mM triethanolamine buffer, pH 7.5, and passed through aMICROFLUIDIZER® high pressure homogenizer (Microfluidics), with standardE. coli lysis settings and maintained at 4° C. Cell debris was removedby centrifugation (10,000 rpm, 45 minutes, 4° C.). The cleared lysatesupernatant was collected and frozen at −80° C. and then eitherHis-affinity purified and dialyzed to produce purified protein orlyophilized to produce a dry shake-flask powder of crude protein.

Assay of SFP for Stevioside Glucosylation:

SFP was reconstituted to provide 20 g/L powder. Then, 50 μL of thesestocks were diluted in 200 μL total reaction volume of 50 mM Tris-HClbuffer, pH 7.5, with 3 mM MgSO4 and 1 mM stevioside (ChromaDex, >94%purity), with 2 mM uridine diphosphoglucose. The reaction was performedat 30° C. in a Thermotron® titre-plate shaker with 300 RPM shaking for16-18h.

HPLC-MS/MS Analysis:

The reaction described above was quenched with 0.5 volume/volumeacetonitrile with 0.2% formic acid and precipitated by centrifugation.Glycosylated stevioside products were detected in the supernatant byLC-MS/MS with the following instrument and parameters:

TABLE 1.1 HPLC-MS/MS Analysis of Steviol Glycosides Instrument AgilentHPLC 1200 series, Sciex 4000 QTrap Column Poroshell 120 EC C18 50 × 3.0mm, 2.7 μm with Poroshell 120 EC C18 5 × 3.0, 2.7 μm guard column(Agilent Technologies) Mobile phase Gradient (A: 0.1% formic acid inwater, B: 0.1% formic acid in methanol) Time (m) % B 0 60 0.50 60 1.0070 4.33 70 5.00 95 5.33 9 5.34 60 6.00 60 Flow rate 0.8 mL/m Run time 6m Peak retention times Rebaudioside A: 2.35 m Column temperature 40° C.Injection volume 10 μL MS detection MRM 990/828 (for stevioltetraglycosides, e.g., rebaudioside A), 1152/828 (for steviolpentaglycosides, e.g., rebaudioside D), 1314/828 (steviolhexaglycosides, e.g., rebaudioside M), 828/666 (for stevioltriglycosides, e.g., stevioside), 666/504 (steviol diglycosides, e.g.,rubusoside) MS conditions MODE: MRM; CUR: 30; IS: 4750; CAD: high; TEM:550° C.; GS1: 50; G52: 50; DP: 150; EP: 10; CXP: 14; DT: 50 ms for eachtransition. For the first three transitions CE: 85; for the last twotransitions CE: 60.

Activity was detected for SEQ ID NO:4. High conversion (i.e., >95%), ofstevioside to rebaudioside A was observed in the LC-MS/MS analysis ofthe assay samples described above.

Example 2 GT Variants of SEQ ID NO: 4

In this Example, experiments for evolution and screening of GTpolypeptides derived from SEQ ID NO: 4 for improved glucosylation ofsteviol glycosides using ADP-glucose are described. Directed evolutionof the GT encoded by SEQ ID NO: 3 (i.e., SEQ ID NO:4) was carried out byconstructing libraries of variant genes in which positions associatedwith certain structural features of the enzyme were subjected tomutagenesis. These libraries were then plated, grown, and screened usingthe high-throughput (HTP) assay described below to provide a first round(“Round 1”) of 12 engineered GT variant polypeptides withglucosyltransferase activity toward ADP-glucose and steviol glycosides.

HTP Growth, Expression, and Lysate Preparation

Cells were picked into 96-well plates and grown overnight in LB mediacontaining 1% glucose and 30 μg/mL CAM, 30° C., 200 rpm, 85% humidity.Then, 20 μL of overnight growth were transferred to a deep-well platecontaining 380 μL TB growth media containing 30 μg/mL CAM, induced with1 mM IPTG, and incubated for 18 h at 30° C., 200 rpm, 85% humidity. Cellcultures were centrifuged at 4000 rpm, 4° C. for 10 m, and the mediadiscarded. Cell pellets thus obtained were frozen at −80° C., and lysedin 250 μL lysis buffer (0.5 g/L lysozyme and 0.5 g/L PMBS in 20 mMTris-HCl buffer, pH 7.5) with low-speed shaking for 2 h on titre-plateshaker at room temperature. The plates were then centrifuged at 4000 rpmand 4° C. for 20 min and the cleared lysate supernatants were used inthe HTP assay reactions described below.

HTP Assay for Glucose Transfer from ADP-Glucose to Stevioside:

Assays were performed on 96-well plates of cleared E. coli culturelysates expressing SEQ ID NO: 3 variants with lysate loading of 50 μLlysate in 200 μL reactions and with substrate loading of 1 mM stevioside(ChromaDex, >94% purity), from a 20 mM stock solution in 50% ethanol andco-substrate loading of 0.5 mM ADP-glucose (Sigma, >93% purity). Thefollowing reaction conditions were used: 50 mM Tris-HCl buffer, pH 7.5,3 mM MgCl₂, 30° C. in a Thermotron® titre-plate shaker with 300 RPMshaking for 18 h. The reactions were quenched with 100 μL/wellacetonitrile with 0.2% formic acid, centrifuged 10 m at 4° C., and thesupernatants were analyzed by HPLC-MS/MS as described in Example 1,Table 1.1.

Formation of rebaudioside A from stevioside in the presence of wild-typeUGT76G1 (SEQ ID NO:4) with ADP-glucose was indistinguishable from a noenzyme control. In contrast to the wild-type enzyme of SEQ ID NO:4,glycosyltransferase variant polypeptides were identified that producedrebaudioside A from stevioside with ADP-glucose. The engineeredpolypeptides are listed in Table 2.1. Although the parent and variantconstructs contain an N-terminal histidine tag for affinitypurification, the mutations were numbered relative to the untaggedreference sequence for clarity. Shake-flask scale cultures were grownfor protein purification as described in Example 1. Variants with thefollowing amino acid mutations shown in Table 2.1, relative to SEQ IDNO:4 were analyzed.

TABLE 2.1 Variants and RebA Levels SEQ ID NO: Amino Acid DifferencesIncreased (nt/aa) (Relative to SEQ ID NO: 4) RebA^(a) 5/6 V309R ++ 7/8R10-/V309R ++  9/10 S361G ++ 11/12 V309S ++ 13/14 L307V + 15/16 S283T +17/18 V309L + 19/20 Y278L/T284I/R311G/V339A/N360G + 21/22 V309N + 23/24R262L + 25/26 V339A/S361G + 27/28 V344I/S361G + 29/30 R262K + ^(a)Levelsof increased production were determined relative to the referencepolypeptide of SEQ ID NO: 4, and defined as follows: “+” = production atleast that of the reference polypeptide, but less than 1.3-fold; and“++” = at least 1.3-fold, but less than 1.6-fold increased production,as compared to the reference polypeptide.Purified Protein Characterization Assay and Analysis for GlucosylTransfer from ADP-Glucose to Stevioside or Rebaudioside D:

First, 250 mL shake flask cultures were grown, induced, lysed,histidine-affinity purified, dialyzed, and diluted 1:1 with glycerol asdescribed in Example 1, to produce purified protein. Then, 50 μL ofthese proteins were diluted in 200 μL total reaction volume of 50 mMTris-HCl buffer, pH 7.5, 3 mM MgCl₂, with 1 mM stevioside(ChromaDex, >94% purity) or rebaudioside D (Sigma, >93% purity) and 0.5mM ADP-glucose. The reaction was performed at 30° C. in a Thermotron®titre-plate shaker with 300 RPM shaking for 16 h. The reaction describedabove was quenched with 0.5 volume/volume acetonitrile with 0.2% formicacid and precipitated by centrifugation. Glycosylated products weredetected in the supernatant by LC-MS/MS following 1:10 dilution in waterwith the instrument and parameters described in Example 1, Table 1.1.

TABLE 2.2 Purified Round 1 Variants and RebA and RebM Levels SEQ ID NO:Amino Acid Differences Increased Increased (nt/aa) (Relative to SEQ IDNO: 4) RebA^(a) RebM^(a) 7/8 R10-/V309R +++ +  9/10 S361G ++ − 11/12V309S ++ + 13/14 L307V ++ − 15/16 S283T +++ − 29/30 R262K − − ^(a)Levelsof increased production were determined relative to the referencepolypeptide of SEQ ID NO: 4, and defined as follows: “−“ = productionless than 1.5-fold; “+” = production at least 1.5- fold, but less than2.5-fold; “++” = at least 2.5-fold, but less than 3.5-fold; and “+++” =at least 3.5-fold increased production, as compared to the referencepolypeptide.

Variants with mutations R10- and V309R, S361G, V309S, L307V, and S283T(SEQ ID NOS: 8, 10, 12, 14, and 16) produced rebaudioside A fromstevioside, at levels above the above negative control and SEQ ID NO: 2levels with ADP-glucose. The variant with mutations R10- and V309R (SEQID NO: 8) and the variant with mutation V309S (SEQ ID NO: 12) producedrebaudioside M from rebaudioside D above negative control and SEQ ID NO:2 levels with ADP-glucose. Thus, these engineeredADP-glycosyltransferase enzymes provide new biocatalytic reagents foruse in new methods for the β-glucosylation of stevioside to rebaudiosideA, and rebaudioside D to rebaudioside M. The variant with mutations R10-and V309R (SEQ ID NO: 8) had the highest activity on both stevioside andrebaudioside D with ADP-glucose as a co-substrate. Thus, the encodingpolynucleotide (SEQ ID NO: 7) was selected for further directedevolution.

Example 3 ADP-Glycosyltransferase Variants of SEQ ID NO: 8

In this Example, experiments for evolution and screening of GTpolypeptides derived from SEQ ID NO: 8, for improved glucosylation ofsteviol glycosides using ADP-glucose are described. Directed evolutionof the GT encoded by SEQ ID NO: 7 (i.e., SEQ ID NO:8) was carried out byconstructing libraries of variant genes in which mutations associatedwith improved activity in Round 1 were recombined. These libraries werethen plated, grown, and screened using the high-throughput (HTP) assaydescribed in Example 2 to provide a second round (“Round 2”) of 20engineered GT variant polypeptides with glucosyltransferase activitytoward ADP-glucose and steviol glycosides. The engineered polypeptidesare listed in Table 3.1. Although the parent and variant constructscontain an N-terminal histidine tag for affinity purification, themutations were numbered relative to the untagged reference sequence forclarity. Shake-flask scale cultures were grown for protein purificationas described in Example 1 for variants with the amino acid mutationsshown in Table 3.1, relative to SEQ ID NO:8, as indicated below, wereanalyzed.

TABLE 3.1 Variants and RebA Levels SEQ ID NO: Amino Acid DifferencesIncreased (nt/aa) (Relative to SEQ ID NO: 8) RebA^(a) 31/32T283Q/T318E/W337S/S360G +++ 33/34 E112S/T172S/T283Q/T318E ++ 35/36 T283Q++ 37/38 T283Q/S360G ++ 39/40 T283Q/L306V/R308S/S360G + 41/42S282T/T283Q/Q431E + 43/44 N137K/T283Q + 45/46 T318E + 47/48E112S/R261S/T318E + 49/50 N137K/T283Q/Q431E + 51/52 S282T/T283Q + 53/54L163K/T318E + 55/56 T283Q/L306V/W337S/A426V + 57/58 R261S/T283Q/W337F +59/60 E112S/S282T/T283Q/Q431E + 61/62 R261S/W337S + 63/64 Q431E + 65/66S360G + 67/68 Q269T/T318E + 69/70 R261S/T283Q/L306V/W337F + ^(a)Levelsof increased production were determined relative to the referencepolypeptide of SEQ ID NO: 7, and defined as follows: “+” = production atleast that of the reference polypeptide, but less than 1.5-fold; “++” =at least 1.5-fold, but less than 2-fold; and “+++” = at least 2-foldincreased production, as compared to the reference polypeptide.Purified Protein Characterization Assay and Analysis for GlucosylTransfer from ADP-Glucose to Stevioside or Rebaudioside D

Proteins were purified, assayed, and analyzed as described in Example 2.

TABLE 3.2 Purified Round 2 Variants and RebA and RebM Levels SEQ ID NO:Amino Acid Differences Increased Increased (nt/aa) (Relative to SEQ IDNO: 8) RebA^(a) RebM^(a) 31/32 T283Q/T318E/W337S/S360G +++ +++ 33/34E112S/T172S/T283Q/T318E + ++ 35/36 T283Q + + 37/38 T283Q/S360G + ++^(a)Levels of increased production were determined relative to thereference polypeptide of SEQ ID NO: 7, and defined as follows: “+” =production at least that of the reference polypeptide, but less than2-fold; “++” = at least 2-fold, but less than 3-fold; and “+++” = atleast 3-fold increased production, as compared to the referencepolypeptide.

All variants in Table 3.2 (SEQ ID NOS: 32, 34, 36, and 38) producedrebaudioside A from stevioside and rebaudioside M from rebaudioside Dwith ADP-glucose, at greater quantities than SEQ ID NO: 8. The variantwith mutations T283Q, T318E, W337S, and S360G (SEQ ID NO: 32) had thehighest activity on both stevioside and rebaudioside D with ADP-glucoseas a co-substrate. Thus, the encoding polynucleotide (SEQ ID NO: 31) wasselected for further directed evolution.

Purified Protein Characterization Assay and Analysis for GlucosylTransfer from NDP-Glucose to Stevioside

To profile the nucleotide diphosphate specificity of SEQ ID NO: 4relative to SEQ ID NO: 32, the following experiment was performed.First, 50 μL purified protein was diluted in 200 μL total reactionvolume of 50 mM Tris-HCl buffer, pH 7.5, 3 mM MgCl₂, with 1 mMstevioside (ChromaDex, >94% purity) and 0.5 mM ADP-glucose, UDP-glucose,or TDP-glucose. The reaction was performed at 30° C. in a Thermotron®titre-plate shaker with 300 RPM shaking for 16 h. The reaction describedabove was quenched with 0.5 volume/volume acetonitrile with 0.2% formicacid and precipitated by centrifugation. Glycosylated products weredetected in the supernatant by LC-MS/MS following 1:10 dilution in waterwith the instrument and parameters described in Example 1, Table 1.1.SEQ ID NO: 32 produced 13-fold more rebaudioside A with ADP-glucose thandid SEQ ID NO: 4, 90% as much with UDP-glucose, and 22% as much withTDP-glucose. The glycosyltransferase encoded by SEQ ID NO: 31 (i.e., SEQID NO:32) has substantially modified NDP-glucose specificity, ascompared to the glycosyltransferase encoded by SEQ ID NO: 3 (i.e., SEQID NO:4).

Determination of Specific Activity of GT Encoded by SEQ ID NO: 31 onRebaudioside D and NDP-Glucose

To profile the nucleotide diphosphate specificity of SEQ ID NO: 32, thefollowing experiment was performed. First, 5 μL purified protein wasdiluted in 100 μL total reaction volume of 50 mM Tris-HCl buffer, pH7.5, 3 mM MgCl₂, with 1 mM rebaudioside D (ChromaDex, >93% purity) and 2mM ADP-glucose, UDP-glucose, TDP-glucose, or GDP-glucose. The reactionwas performed at 30° C. in a Thermotron® titre-plate shaker with 300 RPMshaking for 1-18 h. The reaction described above was quenched with 0.5volume/volume acetonitrile with 0.2% formic acid and precipitated bycentrifugation. Glycosylated products were detected in the supernatantby LC-MS/MS, following 1:10 dilution in water with the instrument andparameters described in Example 1, Table 1.1. The glycosyltransferaseencoded by SEQ ID NO: 31 (i.e., SEQ ID NO:32), produced 50% as muchrebaudioside M with GDP-glucose relative to ADP-glucose, 70% as muchwith UDP-glucose, and an amount below the detection limit withTDP-glucose. The specific activity (μmol RebM formed per mg purifiedprotein per min) of GT SEQ ID NO: 32 with rebaudioside D and ADP-glucosewas 1.4-fold higher than the specific activity with UDP-glucose.Therefore, the glycosyltransferase encoded by SEQ ID NO: 31 (i.e., SEQID NO:32) is a new adenine diphosphoglucose-dependentglycosyltransferase, or “AGT.”

Example 4 Transformation of Stevioside to Rebaudioside A with EngineeredAGT SEQ ID NO: 32 and In Situ Formation of ADP-Glucose

In this Example, experiments to assess the in situ formation ofADP-glucose for glucosylation of steviol glycosides (See, FIG. 1) aredescribed.

Gene Synthesis and Optimization

The polynucleotide sequences encoding the wild-type Acidithiobacilluscaldus sucrose synthase and Thermosynechococcus elongatus sucrosesynthase polypeptides (SEQ ID NOS: 72 and 74, respectively) reported topreferentially utilize ADP-glucose to donate a glucose to fructose toform sucrose in a reversible conversion (See e.g., Diricks et al., Appl.Microbiol. Biotechnol., 99:8465-74 [2015] and Figueroa et al., FEBSLett., 587: 165-9 [2013]), were codon-optimized and synthesized as thegene of SEQ ID NOS: 71 and 73. These synthetic genes (SEQ ID NOS: 71 and73) were individually cloned into a pCK110900 vector system (See e.g.,US Pat. Appln. Pubn. No. 2006/0195947, which is hereby incorporated byreference herein), and subsequently expressed in E. coli W3110 (ΔfhuA).The E. coli strain W3110 expressed the enzymes under the control of thelac promoter. The strains were grown at shake-flask scale and lysed forHis-affinity protein purification, which was performed as described inExample 1.

Coupled Assay with Purified AGT and Sucrose Synthase

To examine the potential for NDP recycling by coupling a sucrosesynthase (SuS) with the AGT encoded by SEQ ID NO: 31 (i.e., SEQ IDNO:32), the following experiment was performed. First, 20 μL purifiedsucrose synthase polypeptide (SEQ ID NO: 72 or 74) and 30 μL purifiedAGT polypeptide (SEQ ID NO: 32) were diluted in 200 μL total reactionvolume of 50 mM Tris-HCl buffer, pH 7.5, 5 mM MgCl₂, with 1 mMstevioside (ChromaDex, >94% purity), 200 mM sucrose, and 5 mM adenosinediphosphate (ADP), cytidine diphosphate (CDP), guanosine diphosphate(GDP), thymidine diphosphate (TDP), or uridine diphosphate (UDP) (Sigma,all 5 >93% purity). The reaction was performed at 30° C. in aThermotron® titre-plate shaker with 300 RPM shaking for 16 h. Thereaction described above was quenched with 0.5 volume/volumeacetonitrile with 0.2% formic acid and precipitated by centrifugation.

Glycosylated products were detected in the supernatant by LC-MS/MSfollowing 1:10 dilution in water with the instrument and parametersdescribed in Example 1, Table 1.1.

For SEQ ID NO: 32 and SuS (SEQ ID NO: 74), comparable amounts ofrebaudioside A were formed with ADP, UDP, and GDP, and almost norebaudioside A was formed with CDP and TDP. For SEQ ID NO: 32 and SuS(SEQ ID NO: 72), the level of rebaudioside A formed with ADP wascomparable to that formed with SuS (SEQ ID NO: 74) with ADP. The amountof rebaudioside A formed with UDP was less than 20% of that formed withADP, and almost no rebaudioside A was formed with GT (SEQ ID NO: 32) andSuS (SEQ ID NO: 72) and CDP, GDP, or TDP. These results demonstrate thatboth SEQ ID NOS: 72 and 74 are capable of generating ADP-glucose in situfrom sucrose and find use with AGT (SEQ ID NO: 32) for glucosylation ofsteviol glycosides with sucrose and ADP as co-substrates instead of themore expensive substrate ADP-glucose. In addition, SEQ ID NO: 72 canalso be used with GDP and UDP. In some embodiments involvingADP-selective coupling systems, SEQ ID NO: 74 finds use.

Example 5 ADP-Glycosyltransferase Circular Permuted Variants of SEQ IDNO: 32

In this Example, experiments for design, construction, and evaluation ofGT polypeptides derived from SEQ ID NO: 32 for glucosylation of steviolglycosides using ADP-glucose are described. Directed evolution of the GTencoded by SEQ ID NO: 31 was carried out by constructing libraries ofvariant genes in which the N- and C-termini were linked in the encodingsequence and positions associated with certain structural features ofthe enzyme were selected as the new N-terminus of the protein. Thislibrary of circular permuted variants was then plated, grown, andscreened using the high-throughput (HTP) assay described below toprovide a “Round 3.2” of 17 engineered GT circular permuted variantpolypeptides with glucosyltransferase activity toward ADP-glucose andsteviol glycosides. None of the variants had activity higher than theunpermuted SEQ ID NO: 32, but the 17 engineered polypeptides listed inTable 5.1 had activity higher than a no enzyme negative control.

HTP Assay for Glucose Transfer from ADP-Glucose to Stevioside

Assays were performed on 96-well plates of cleared E. coli culturelysates expressing SEQ ID NO: 31 variants with lysate loading of 25 μLlysate in 200 μL reactions and with substrate loading of 1 mM stevioside(ChromaDex, >94% purity), from a 20 mM stock solution in 50% ethanol andco-substrate loading of 0.5 mM ADP-glucose (Sigma, >93% purity). Thefollowing reaction conditions were used: 50 mM Tris-HCl buffer, pH 7.5,3 mM MgCl₂, 30° C. in a Thermotron® titre-plate shaker with 300 RPMshaking for 4 h. The reactions were quenched with 100 μL/wellacetonitrile with 0.2% formic acid, centrifuged 10 m at 4° C., and thesupernatants were analyzed by HPLC-MS/MS as described in Example 1,Table 1.1.

The engineered polypeptides analyzed are listed in Table 5.1. Althoughthe parent and variant constructs contain an N-terminal histidine tagfor affinity purification, the amino acids were numbered relative to theuntagged reference sequence for clarity. Shake-flask scale cultures weregrown for protein purification as described in Example 1, for variantswith the following first amino acids relative to SEQ ID NO 32: 71, 170,259, and 401. These variants represented circular permutants from themost distinct regions of the protein.

TABLE 5.1 Circular Permuted Round 3.2 Variants SEQ ID NO: First AminoAcid Position (nt/aa) (Relative to SEQ ID NO: 32) 75/76 70 77/78 7179/80 72 81/82 73 83/84 74 85/86 75 87/88 169 89/90 170 91/92 171 93/94174 95/96 194 97/98 198  99/100 259 101/102 260 103/104 370 105/106 401107/108 403

Determination of Specific Activity of Purified Circular Permuted GTs onRebaudioside D and ADP-Glucose

First, 10 μL purified protein was diluted in 100 μL total reactionvolume of 50 mM Tris-HCl buffer, pH 7.5, 3 mM MgCl₂, with 1 mMrebaudioside D (ChromaDex, >93% purity) and 2 mM ADP-glucose. Thereaction was performed at 30° C. in a Thermotron® titre-plate shakerwith 300 RPM shaking for 1-4 h. The reaction described above wasquenched with 0.5 volume/volume acetonitrile with 0.2% formic acid andprecipitated by centrifugation.

Glycosylated products were detected in the supernatant by LC-MS/MSfollowing 1:10 dilution in water with the instrument and parametersdescribed in Example 1, Table 1.1. Of the four circular permutants, SEQID NO: 106 had the highest specific activity (μmol RebM formed per mgpurified protein per min), followed by SEQ ID NO: 100, and SEQ ID NO:90. SEQ ID NO: 78 had barely detectable activity. Thus, theglycosyltransferase encoded by SEQ ID NO: 105 (i.e., SEQ ID NO:106) wasidentified in these experiments as the best candidate circular permutedAGT for further directed evolution.

Example 6 ADP-Glycosyltransferase Variants of SEQ ID NO: 32

In this Example, experiments for evolution and screening of GTpolypeptides derived from SEQ ID NO: 32 for improved glucosylation ofsteviol glycosides using ADP-glucose are described. Directed evolutionof the GT encoded by SEQ ID NO:31 (i.e., SEQ ID NO:32) was carried outby constructing libraries of variant genes in which positions associatedwith certain structural features of the enzyme were subjected tomutagenesis. These libraries were then plated, grown, and screened usingthe high-throughput (HTP) assay described below to provide a third round(“Round 3.1”) of 60 engineered GT variant polypeptides withglucosyltransferase activity toward ADP-glucose and steviol glycosides.

HTP Assay for Glucose Transfer from ADP-Glucose to Stevioside orRebaudioside D

Assays were performed on 96-well plates of cleared E. coli culturelysates expressing SEQ ID NO: 32 variants with lysate loading of 25 μLlysate in 200 μL reactions and with substrate loading of 0.5 mMrebaudioside D or 1 mM stevioside (ChromaDex, >94% purity), from a 20 mMstock solution in 50% ethanol and co-substrate loading of 0.5 mMADP-glucose (Sigma, >93% purity). The following reaction conditions wereused: 50 mM Tris-HCl buffer, pH 7.5, 3 mM MgCl₂, 30° C. in a Thermotron®titre-plate shaker with 300 RPM shaking for 4h. The reactions werequenched with 100 μL/well acetonitrile with 0.2% formic acid,centrifuged 10 mat 4° C., and the supernatants were analyzed byHPLC-MS/MS as described in Example 1, Table 1.1.

The engineered polypeptides are listed in Table 6.1. Although the parentand variant constructs contain an N-terminal histidine tag for affinitypurification, the mutations were numbered relative to the untaggedreference sequence for clarity. Shake-flask scale cultures were grownfor protein purification as described in Example 1 for variants with theamino acid mutations shown in Table 6.1, relative to SEQ ID NO: 32.

TABLE 6.1 Round 3.1 Variants and RebA/RebM Levels SEQ ID NO: Amino AcidDifferences Increased Increased (nt/aa) (Relative to SEQ ID NO: 32)RebA^(a) RebM^(a) 109/110 L84A + 111/112 L199G + 113/114 L84G +++115/116 M87A + 117/118 L199Q + 119/120 L199D + 121/122 I207L + 123/124M87H ++ 125/126 I198M + 127/128 L199S +++ 129/130 H154Q + 131/132S191R + 133/134 I198S + 135/136 L199K +++ 137/138 H154L +++ 139/140L199A +++ 141/142 H154V ++ 143/144 I198V + 145/146 H154A + 147/148 Q22A++ 149/150 Q22L + 151/152 Q22P +++ 153/154 S356G +++ 155/156 Q22H +157/158 L306V + 159/160 T262G + 161/162 D169T + 163/164 G347D + 165/166S179V + 167/168 Q159M + 169/170 I233R + 171/172 Y396R ++ 173/174 E6P +175/176 R139P ++ 177/178 Y421V + 179/180 R261W + 181/182 L106S ++183/184 N137G ++ 185/186 A97S ++ 187/188 R74W + 189/190 A110S + 191/192R261P +++ 193/194 Q159R + 195/196 N195G + 197/198 E417R + 199/200L106T + 201/202 R427A + 203/204 F64P + 205/206 H259Q + 207/208 R261H +209/210 E417P + 211/212 K4P + 213/214 V435Q + 215/216 L106G + 217/218L106D + 219/220 R261A + 221/222 E417A + 223/224 E112P + 225/226 V435R +227/228 E112A + ^(a)Levels of increased production were determinedrelative to the reference polypeptide of SEQ ID NO: 32, and defined asfollows: “−“ = production less than the reference polypeptide; “+” =production at least that of the reference polypeptide, but less than2-fold; “++” = at least 2-fold, but less than 3-fold; and “+++” = atleast 3-fold increased production, as compared to the referencepolypeptide.Purified Protein Characterization Assay and Analysis for GlucosylTransfer from ADP-Glucose to Stevioside or Rebaudiosided D

First, 1 μL purified protein was diluted in 100 μL total reaction volumeof 50 mM Tris-HCl buffer, pH 7.5, 3 mM MgCl₂, with 1 mM stevioside(Chromadex, >94% purity) or rebaudioside D (ChromaDex, >93% purity) andmM ADP-glucose. The reaction was performed at 30° C. in a Thermotron®titre-plate shaker with 300 RPM shaking for 1-30 h. The reactiondescribed above was quenched with 0.5 volume/volume acetonitrile with0.2% formic acid and precipitated by centrifugation. Glycosylatedproducts were detected in the supernatant by LC-MS/MS following 1:10dilution in water with the instrument and parameters described inExample 1, Table 1.1. Specific activities described in Table 6.2 forstevioside and rebaudioside D were determined as μmol product formed permin per mg purified protein from the linear portion of the reactionprogress curve.

TABLE 6.2 Round 3.1 Variant Specific Activity for Stevioside and RebDSEQ ID NO: Amino Acid Differences μmol RebA/ μmol RebM/ (nt/aa)(Relative to SEQ ID NO: 32) min-mg^(a) min-mg^(a) 113/114 L84G + ++123/124 M87H − − 127/128 L199S − ++ 151/152 Q22P + +++ 153/154 S356G ++++ 157/158 L306V − ++ ^(a)Levels of increased specific activity weredetermined relative to the reference polypeptide of SEQ ID NO: 32, anddefined as follows: “−“ = activity less than 1.5-fold; “+” = activity atleast 1.5-fold, but less than 3-fold; “++” = at least 3-fold, but lessthan 6-fold; and “+++” = at least 6-fold increased activity, as comparedto the reference polypeptide.

Directed evolution of the GT encoded by SEQ ID NO:31 (i.e., SEQ IDNO:32) was continued by constructing libraries of variant genes in whichmutations associated with improved activity above were recombined. Theselibraries were then plated, grown, and screened using thehigh-throughput (HTP) assay described above with rebaudioside D toprovide Round 3.3 with 59 engineered GT variant polypeptides withglucosyltransferase activity toward ADP-glucose and steviol glycosides.The engineered polypeptides are listed in Table 6.3. Shake-flask scalecultures were grown for protein purification as described in Example 1for variants with the amino acid mutations as shown in Table 6.3,relative to SEQ ID NO: 32.

TABLE 6.3 Round 3.3 Variants and RebM Levels SEQ ID NO: Amino AcidDifferences Increased (nt/aa) (Relative to SEQ ID NO: 32) RebM^(a)229/230 Q22P/L84G/H154L/I198S/L199A/S356G ++++ 231/232Q22P/L84G/H154L/L199A/S356G ++++ 233/234Q22P/L84G/H154L/I198S/L199S/S356G ++++ 235/236Q22P/L84V/H154V/I198S/L199S/L306V/ ++++ S356G 237/238Q22P/L84G/H154V/I198S/L199A/L306V/ ++++ S356G 239/240Q22P/L84G/H154L/I198S/L199K/S356G ++++ 241/242Q22P/L84G/H154L/L199S/S356G ++++ 243/244Q22P/L84G/M87H/H154L/I198S/L199S/ ++++ L306V/S356G 245/246Q22P/L84G/H154V/L199A/L306V/S356G ++++ 247/248Q22P/L84G/M87H/H154L/I198S/L199A/ ++++ S356G 249/250Q22P/L84G/H154V/I198S/L199K/L306V/ ++++ S356G 251/252Q22P/L84G/M87H/H154L/L199K/L306V/ ++++ S356G 253/254Q22P/L84G/H154V/L199S/S356G ++++ 255/256Q22P/M87H/H154L/L199A/L306V/S356G +++ 257/258Q22P/L84G/M87H/H154V/I198S/L199S/ +++ L306V/S356G 259/260Q22P/L84G/I198S/L199K/L306V/S356G +++ 261/262Q22P/L84G/H154V/L199A/S356G +++ 263/264Q22P/H154L/I198S/L199A/I207L/L306V/ +++ S356G 265/266Q22P/H154L/L199A/S356G +++ 267/268 Q22P/H154L/I198S/L199A/S356G +++269/270 Q22P/L84G/M87H/H154V/L199K/L306V/ +++ S356G 271/272Q22P/M87H/H154V/I198S/L199S/I207L/ +++ L306V/S356G 273/274Q22P/L84G/H154L/I207L/L306V/S356G +++ 275/276Q22P/L84G/H154L/L306V/S356G +++ 277/278 Q22P/M87H/H154V/L199S/S356G ++279/280 Q22P/M87H/L199A/S356G ++ 281/282Q22P/H154V/I198S/L199S/L306V/S356G ++ 283/284Q22P/L84G/M87H/L199A/L306V/S356G ++ 285/286Q22P/M87H/H154V/L199A/I207L/S356G ++ 287/288 Q22P/L84G/L199S/S356G ++289/290 Q22P/L84G/H154L/I198S/L199A/I207L/ ++ L306V 291/292Q22P/M87H/H154L/I198S/L199K/S356G ++ 293/294Q22P/L84G/M87H/I198S/L199K/S356G ++ 295/296 Q22P/H154V/L199A/S356G ++297/298 Q22P/M87H/H154V/L199K/S356G ++ 299/300Q22P/H154V/L199K/I207L/L306V/S356G ++ 301/302 Q22P/L84G/I207L/S356G ++303/304 Q22P/M87H/H154V/L199S/L306V/S356G ++ 305/306Q22P/M87H/L199K/S356G ++ 307/308 Q22P/M87H/I198S/L199K/L306V/S356G ++309/310 Q22P/M87H/H154L/P322S/S356G ++ 311/312Q22P/M87H/I198S/L199K/S356G + 313/314 Q22P/L84G/H154L/L199K/I207L +315/316 Q22P/M87H/I198S/L199S/I207L/S356G + 317/318Q22P/L84G/M87H/I198S/L199A/L306V/ + S356G 319/320Q22P/L84G/M87H/I198S/L199S/S356G + 321/322Q22P/L84G/M87H/I198S/L199S/L306V/ + S356G 323/324 Q22P/L199A/S356G +325/326 Q22P/L84G/H154L/I198S/L199A/L306V + 327/328L84G/H154L/I198S/L199K + 329/330 Q22P/L84G/M87H/L199S/S356G + 331/332Q22P/L84G/M87H/H154L/S356G + 333/334Q22P/I198S/L199K/I207L/G329C/S356G + 335/336Q22P/M87H/H154V/I198S/L199K/S356G + 337/338 Q22P/I198S/L199A/S356G +339/340 Q22P/M87H/I198S/L199A/L306V/S356G + 341/342 Q22P/I207L/S356G +343/344 Q22P/L84G/S356G + 345/346 Q22P/L84G/H154V/I198S/L199K +1289/1290b Q22P/S356G − ^(a)Levels of increased production weredetermined relative to the reference polypeptide of SEQ ID NO: 32, anddefined as follows: “−“ = production less than 100-fold; the referencepolypeptide; “+ = production at least 100-fold and less than 300-fold;“++” = at least 300-fold, but less than 600-fold; “+++” = at least600-fold, but less than 900-fold; and “++++” = greater than 900-foldincreased production, as compared to the reference polypeptide. ^(b)Doesnot contain diversity specific to conversion of rebaudioside D torebaudioside M; useful as an engineered biocatalyst to convertstevioside to rebaudioside A.Purified Protein Characterization Assay and Analysis for GlucosylTransfer from ADP-Glucose to Stevioside or Rebaudioside D

First, 2 μL purified protein was diluted in 100 μL total reaction volumeof 50 mM Tris-HCl buffer, pH 7.5, 3 mM MgCl₂, with 1 mM rebaudioside D(ChromaDex, >93% purity) and 2 mM ADP-glucose. The reaction wasperformed at 30° C. in a Thermotron® titre-plate shaker with 300 RPMshaking for 1-18 h. The reaction described above was quenched with 0.5volume/volume acetonitrile with 0.2% formic acid and precipitated bycentrifugation.

Glycosylated products were detected in the supernatant by LC-MS/MSfollowing 1:50 dilution in water with the instrument and parametersdescribed in Example 1, Table 1.1. Specific activities described inTable 6.4 for stevioside was determined as mol rebaudioside A productformed per min per mg purified protein and for rebaudioside D wasdetermined as μmol rebaudioside M product formed per min per mg purifiedprotein from the linear portion of the reaction progress curve. Theenzymes listed in Table 6.4 catalyzed the conversion of RebD to RebMto >99% conversion and catalyzed the conversion of stevioside to amixture of RebA and RebI with >85% conversion in less than 18 h with 0.8g/L stevioside or 1.3 g/L rebD, 2 molar excess of ADP-glucose, and 35-77mg/L purified protein.

TABLE 6.4 Round 3.3 Variants Specific Activity for Stevioside and RebDμmol μmol SEQ ID NO: Amino Acid Differences RebA/ RebM/ (nt/aa)(Relative to SEQ ID NO: 32) min-mg^(a) min-mg^(a) 229/230Q22P/L84G/H154L/I198S/L199A/ + ++ S356G 231/232Q22P/L84G/H154L/L199A/S356G ++ +++ 233/234 Q22P/L84G/H154L/I198S/L199S/++ ++ S356G 237/238 Q22P/L84G/H154V/I198S/L199A/ ++ ++ L306V/S356G243/244 Q22P/L84G/M87H/H154L/I198S/ + + L199S/L306V/S356G 253/254Q22P/L84G/H154V/L199S/S356G +++ + ^(a)Levels of increased specificactivity were determined relative to the reference polypeptide of SEQ IDNO: 32, and defined as follows: “+” = activity at least that of thereference polypeptide, but less than 2-fold; “++” = at least 2-fold, butless than 4-fold; and “+++” = at least 4-fold increased activity ascompared to the reference polypeptide. ^(b)Levels of increased activitywere determined relative to the reference polypeptide of SEQ ID NO: 32,and defined as follows: “−“ = activity less than 10-fold; “+” = activityat least 10-fold, but less than 15-fold; “++” = at least 15-fold, butless than 20-fold; and “+++” = at least 20-fold increased activity, ascompared to the reference polypeptide.

Example 7 Transformation of Rebaudioside D to Rebaudioside M with AGT(SEQ ID NO: 232)

A 250-mL shake-flask culture was grown for protein purification of thepolypeptide SEQ ID NO: 232 as described in Example 1. Then, 2.4 mL ofthe 50% glycerol stock of was diluted in 60 mL total reaction volume ina 250-mL baffled flask with 50 mM Tris-HCl buffer, pH 7.5, 3 mM MgCl2, 2mM ADP-glucose, and 1 mM rebaudioside D (ChromaDex, >93% purity). Thereaction was performed at 30° C. in an Innova® shaking incubator with250 RPM shaking for 2 h and quenched to pH<4 with 0.12 mL formic acid.The reaction was precipitated by centrifugation at 10,000 RPM for 10 mat 4° C. 6 g XAD-4 resin (Sigma) were added to the supernatant andincubated in the shake flask for 2 h. The resin was filtered and elutedwith 16.3 mL 50:24:26 water:ACN:EtOH by incubating 4 h and re-filtering.A second elution was performed with 10 mL 50:50 water:EtOH, whichfiltered and combined with the first elution. The eluent wasconcentrated to about 6 mL by rotary evaporation, filtered throughWHATMAN® UNIPREP® syringeless filters, and fractionated by HPLC usingthe instrument and parameters described in Table 7.1. From the C18column, fractions were manually collected at retention times 5.8-6.2 m.Fractions were pooled, concentrated by rotary evaporation, andlyophilized. The sample was then resuspended in 1.5 mL ethanol andincubated on a stir plate at 80° C. for 2 h, concentrated by rotaryevaporation, and dried 14 h under vacuum at 40° C. The samples wereresuspended in pyridine-d5 and used for 1H, COSY, and HSQC NMR spectraacquisition performed by Acorn NMR.

TABLE 7.1 Semi-Preparative HPLC Fractionation of Steviol GlycosidesInstrument Agilent HPLC 1200 series Column Higgins C18 250 × 10 mm, 5 μm(Higgins Analytical) Mobile phase Isocratic 68:32 A:B A: 0.1% formicacid in water B: 0.1% formic acid in methanol Flow rate 2.5 mL/m Runtime 10 m Peak retention times Rebaudioside M 6.00 m Column temperature40° C. Injection volume 100 μL UV detection 210 nm (for steviolglycosides) 254 nm (for organic contaminants)

The isolated product was determined to be rebaudioside M based onidentity of the spectra to that of an authentic rebaudioside M standard(Chromadex, purity 95.6% by HPLC). By ¹H NMR, the exchangeable protonsin the sample were broader than those in the standard and the samplecontained a minor methyl contaminant. Otherwise, the spectra of thesample and standard were identical. Except for minor impurities, theCOSY and HSQC spectra of the sample and standard were identical. Thepresence of six anomeric protons evident from the ¹H and ¹H-¹³C HSQCspectra [δ_(H) 6.44, δ_(H) 5.85, δ_(H) 5.524, δ_(H) 5.518, δ_(H) 5.49,δ_(H) 5.34] confirmed the presence of six sugar units in the structureand were coincident with the standard, indicating β-anomericconformation. The attachment of sugars at the C2′ and C3′ hydroxylpositions is supported by the relatively downfield chemical shift ofH-2′ (δ_(H) 4.54) and H-3′ (δ_(H) 5.15) in sugar I, suggesting a2,3-branched-D-glucotriosyl substituent at C-19 (See FIG. 2 for carbonnumbering). Peak assignments are listed in Table 7.2 and were determinedfrom ¹H, COSY, and HSQC spectra and compared to literature (See e.g.,Prakash et al., Nat. Prod. Comm., 11:1523-6 [2013]).

TABLE 7.2 NMR Spectra of Enzymatically Produced Rebaudioside M in C₅D₅N.Position δ ¹³C, ppm δ ¹H, ppm ¹H Multiplicity (J, Hz) 1 40.3 0.77 t(12.1) 1 40.3 1.79 m 2 19.6 1.37 m 2 19.6 2.27 m 3 38.4 1.04 m 3 38.42.32 d (13.0) 4 Null 5 57.3 1.08 d (13.9) 6 23.5 2.26 m 6 23.5 2.44 q(12.6) 7 42.6 1.45 m 7 42.6 1.83 m 8 Null 9 54.3 0.93 d (8.49) 10 Null11 20.2 1.67 m 11 20.2 1.79 m 12 38.5 1.88 m 12 38.5 2.78 m 13 Null 1443.3 2.05 m 14 43.3 2.77 m 15 46.5 1.9 d (17) 15 46.5 2.06 m 16 Null 17104.9 4.92 s 17 104.9 5.72 s 18 28.2 1.32 s 19 Null 20 16.7 1.41 s 1′94.9 6.44 d (8.2) 2′ 77 4.55 m 3′ 88.7 5.15 t (8.5) 4′ 70.1 4.22 m 5′78.6 4.15 m 6′ 61.7 4.22 m 6′ 61.7 4.35 m 1″ 96.3 5.52 d (7.8) 2″ 81.54.15 m 3″ 88 5.01 obscured by water 4″ 70.3 4.1 m 5″ 77.7 3.98 m 6″ 62.54.23 m 6″ 62.5 4.35 m 1′′′ 104.7 5.52 d (7.6) 2′′′ 75.8 4.2 m 3′′′ 78.64.17 m 4′′′ 73.2 4.01 m 5′′′ 77.5 3.76 ddd (3.1, 6.3, 9.5) 6′′′ 64 4.3 m6′′′ 64 4.53 m 1′′′′ 104 5.49 d (8.0) 2′′′′ 75.6 4 m 3′′′′ 77.7 4.53 m4′′′′ 71.3 4.19 m 5′′′′ 77.9 4.02 m 6′′′′ 62.1 4.24 m 6′′′′ 62.1 4.35 m1′′′′′ 104.2 5.86 d (7.5) 2′′′′′ 75.5 4.24 m 3′′′′′ 78.4 4.24 m 4′′′′′73.6 4.14 m 5′′′′′ 77.8 3.94 ddd (3.0, 6.6, 9.6) 6′′′′′ 64 4.36 m 6′′′′′64 4.65 d (9.1) 1′′′′′′ 104.2 5.34 d (8.1) 2′′′′′′ 75.5 3.98 m 3′′′′′′78 4.4 m 4′′′′′′ 71.1 4.15 m 5′′′′′′ 78.1 3.88 m 6′′′′′′ 62 4.14 m6′′′′′′ 62 4.35 m

Example 8 ADP-Glycosyltransferase Variants of SEQ ID NO: 232

In this Example, experiments for evolution and screening of GTpolypeptides derived from SEQ ID NO: 232 for improved glucosylation ofsteviol glycosides using ADP-glucose are described. Directed evolutionof the GT encoded by SEQ ID NO:231 (i.e., SEQ ID NO:232) was carried outby constructing a library in which surface residue mutations associatedwith improved activity in round 3 were recombined. This library was thenplated, grown, and screened using the high-throughput (HTP) assaydescribed below to provide a fourth round (“Round 4”) of 76 engineeredGT variant polypeptides with glucosyltransferase activity towardADP-glucose and steviol glycosides.

HTP Assay for Glucose Transfer from ADP-Glucose to Rebaudioside D

Assays were performed on 96-well plates of cleared E. coli culturelysates expressing SEQ ID NO: 232 variants with lysate loading of 2.5 μLlysate in 100 μL reactions and with substrate loading of 0.5 mMrebaudioside D (ChromaDex, >94% purity), from a 20 mM stock solution in50% ethanol and co-substrate loading of 0.5 mM ADP-glucose (Sigma, >93%purity). The following reaction conditions were used: 50 mM Tris-HClbuffer, pH 7.5, 3 mM MgCl₂, 30° C. in a Thermotron® titre-plate shakerwith 300 RPM shaking for 1 h. The reactions were quenched by dilutingthe assay 1:10 and then quenching with 50 μL/well acetonitrile with 0.2%formic acid, centrifuged 10 m at 4° C., and the supernatants werediluted 1:10 in water and analyzed by HPLC-MS/MS as described in Example1, Table 1.1.

The engineered polypeptides are listed in Table 8.1. Although the parentand variant constructs contain an N-terminal histidine tag for affinitypurification, the mutations were numbered relative to the untaggedreference sequence for clarity. Shake-flask scale cultures were grown,lysed, and lyophilized to powder as described in Example 1 for variantswith SEQ ID NOS: 348, 350, 352, 354, 356, 364, 408, and 428.

TABLE 8.1 Round 4 Variants and RebM Levels SEQ ID NO: Amino AcidDifferences Increased (nt/aa) (Relative to SEQ ID NO: 232) RebM^(a)347/348 L109R/S131P/R139P/S179V/R261P/Y396R/ +++ Y421V 349/350L106T/L109R/R139P/G347D/E417A/ +++ Y421V/R427A 351/352L109R/E112P/S179V/G204D/I233R/E417A/ +++ Y421V/R427L 353/354F64P/L109R/E112P/R139P/E417A ++ 355/356L106S/L109R/E112P/S131P/Q159R/G204D/ ++ G347D/E417A/Y421V/R427L 357/358L109R/E112P/R139P/S179V/E417R/R427A ++ 359/360F64P/L106S/R139P/S179L/I233R/E417A/ ++ R427A/Q431D 361/362F64P/L106S/L109R/R139P/S179L/E417R/ ++ Y421V/R427L/Q431D 363/364L106S/L109R/E112P/S131P/Q159R/S179L/ ++ E417A/Y421V 365/366F64P/L109R/R139P/I233R/E417R/Y421V ++ 367/368L106S/E112P/Q159R/G204D/I233R/E417R/ ++ Y421V/R427A/Q431D 369/370L106S/L109R/E112P/R261P/E417R/Q431D ++ 371/372 L106S/E417A ++ 373/374L109R/E112P/Q159R/S179V/E417R/Y421V ++ 375/376L106T/G347D/E417R/R427A/Q431D ++ 377/378F64P/L109R/E112P/R139P/Q159R/S179L/ ++ E417R/Q431D 379/380E112P/S131P/S179L/G347D/E417R/Y421V ++ 381/382F64P/L106T/E112P/Q159R/S179L/E417R/ ++ Y421V 383/384F64P/L106S/L109R/E112P/E417A/Y421V/ + R427A/Q431D 385/386L106S/L109R/E112P/G347D/R427A + 387/388F64P/R139P/G347D/E417R/Y421V/R427L/ + Q431D 389/390F64P/L109R/Q159R/S179V/G204D/I233R/ + E417R/Y421V 391/392F64P/L106T/G204D/E417A/Y421V/R427A + 393/394 S179V/E417R/Y421V + 395/396F64P/L106S/E417R/Y421V + 397/398 E112P/Q159R/E417R/Y421V + 399/400L109R/E112P/E417A/R427A/Q431D + 401/402 L106S/E112P/I233R/E417R +403/404 L109R/E112P/I233R/E417A/Q431D + 405/406L109R/E112P/S131P/Q159R/E417R/Y421V + 407/408L109R/R139P/S179L/E417R/Y421V/R427A + 409/410 L106T/R139P/Y421V +411/412 L109R/E112P/E417A/Y421V/R427L + 413/414 E112P/E417R/Y421V +415/416 F64P/R139P/I233R/E417R/R427L/Q431D/ + K439P 417/418L106T/E112P/Q159R/S179L/G204D/E417A/ + Y421V 419/420I233R/E417R/Y421V/R427L/Q431D + 421/422 L106S/I233R/Y421V/R427A +423/424 L109R/S179V/I233R/Y421V + 425/426F64P/L106T/E112P/R139P/Q159R/S179V/ + G204D/Y396R/E417A 427/428L109R/G204D/E417R/Q431D + 429/430 F64P/L109R/E417R/Y421V + 431/432L109R/E112P/S179V/G347D/E417R + 433/434F64P/L106S/L109R/I233R/G347D/R427A/ + Q431D 435/436L106S/L109R/R139P/R427A/Q431D + 437/438L106S/R139P/Q159R/I233R/G347D/E417R/ + Y421V/R427A/Q431D 439/440L106S/E112P/Y396R/E417R/Y421V + 441/442 L106S/L109R/E112P/G204D/G347D/ +Y421V/K439P 443/444 R139P/I233R/E417A + 445/446 L109R/E417R/Y421V +447/448 L109R/E417R/R427A/Q431D + 449/450 L109R/S131P/G204D + 451/452G347D/E417R + 453/454 L106T/L109R/R139P/E417R + 455/456L106T/L109R/I233R/R427A/Q431D + 457/458 L106S/L109R/E417R/Y421V/R427L +459/460 L109R/E112P/G204D/I233R/E417R + 461/462Q159R/G347D/E417A/Y421V/Q431D + 463/464 L106T/L109R + 465/466L109R/E112P/S131P/Q159R/S179V/K439P + 467/468F64P/L106S/E112P/G347D/E417A/Y421V + 469/470L109R/E112P/Q159R/E417R/R427L + 471/472 L109R/E112P/G204D/R427A +473/474 F64P/L106T/L109R/E112P/S131P/S179L/ + E417R/R427A/Q431D 475/476F64P/L106S/Q431D + 477/478 F64P/E417R/Y421V/Q431D + 479/480L106T/L109R/R139P/I233R/E417R/Y421V + 481/482E112P/S131P/S179V/G204D/E417R/Y421V/ + R427L 483/484L106T/E112P/Q159R/S179V/I233R/E417A/ + Y421V/R427L/K439P 485/486F64P/L106S/L109R/E112P/S131P/Q159R/ + I233R/Y421V/R427L/Q431D 487/488S131P/S179L/I233R/E417R/R427A + 489/490 F64P/L109R/E417A/Y421V + 491/492L109R/R139P/S179L/E417R/R427L + 493/494F64P/L106T/L109R/E417A/Y421V/R427L/ + Q431D/K439P 495/496E112P/R139P/S179V/G204D/I233R/G347D/ + R427L 497/498L106T/S131P/S179L/I233R/Y421V/R427L/ + Q431D ^(a)Levels of increasedproduction were determined relative to the reference polypeptide of SEQID NO: 232, and defined as follows: “+” = production at least 1.6-fold,but less than 2-fold; “++” = at least 2-fold, but less than 2.4-fold;and “+++” = at least 2.4-fold increased production, as compared to thereference polypeptide.Shake Flask Powder Characterization Assay and Analysis for GlucosylTransfer from ADP-Glucose to Stevioside or Rebaudioside D

A time course experiment was performed to characterize the activity ofthe engineered round 4 variants on stevioside and rebaudioside D. Oneg/L shake flask powder (SFP) was added to a 100 μL total reaction volumecontaining 50 mM Tris-HCl buffer, pH 7.5, 3 mM MgCl₂, 2.5% v/v ethanolwith 1 mM stevioside (Chromadex, >94% purity) or rebaudioside D(ChromaDex, >93% purity) and 1 mM ADP-glucose. The reaction wasperformed at 30° C. in a Thermotron® titre-plate shaker with 300 RPMshaking for 0.5-2 h. The reaction was quenched by adding 50 μLacetonitrile with 0.2% formic acid and precipitated by centrifugation.The supernatant was diluted 1:50 in water and analyzed for steviolglycosides by LC-MS/MS as described in Example 1, Table 1.1. All 8variants had higher activities on both stevioside and rebaudioside Dthan SEQ ID NO: 232. The levels of rebaudioside A produced fromstevioside by the variants relative to SEQ ID NO: 232 at the 1 hour timepoint are listed in Table 8.2.

TABLE 8.2 Round 4 Variant SFP Activity on Stevioside SEQ ID NO: AminoAcid Differences Increased (nt/aa) (Relative to SEQ ID NO: 232) RebA^(a)355/356 L106S/L109R/E112P/S131P/Q159R/G204D/ +++ G347D/E417A/Y421V/R427L351/352 L109R/E112P/S179V/G204D/I233R/E417A/ ++ Y421V/R427L 427/428L109R/G204D/E417R/Q431D ++ 347/348 L109R/S131P/R139P/S179V/R261P/Y396R/++ Y421V 363/364 L106S/L109R/E112P/S131P/Q159R/S179L/ ++ E417A/Y421V349/350 L106T/L109R/R139P/G347D/E417A/Y421V/ + R427A 407/408L109R/R139P/S179L/E417R/Y421V/R427A + 353/354F64P/L109R/E112P/R139P/E417A + ^(a)Levels of increased production weredetermined relative to the reference polypeptide of SEQ ID NO: 232, atthe 1 h time point and defined as follows: “+” = production at least1.5-fold, but less than 2-fold; “++” = at least 2-fold, but less than3-fold; and “+++” = at least 3-fold increased production, as compared tothe reference polypeptide.

The activities of the 8 engineered variants were clearly higher onrebaudioside D than the activity of SEQ ID NO: 232, but they were notwell distinguished from each other in the time curse. Therefore, afollow-up experiment was performed as follows: A dose response curve of0.03-1 g/L shake flask powder (SFP) was added to a 100 μL total reactionvolume containing 50 mM Tris-HCl buffer, pH 7.5, 3 mM MgCl₂, 2.5% v/vethanol with 1 mM rebaudioside D (ChromaDex, >93% purity) and 1 mMADP-glucose. The reaction was performed at 30° C. in a Thermotron®titre-plate shaker with 300 RPM shaking for 0.5 h. The reaction wasquenched by adding 50 μL acetonitrile with 0.2% formic acid andprecipitated by centrifugation. The supernatant was diluted 1:67 inwater and analyzed for steviol glycosides by LC-MS/MS as described inExample 1, Table 1.1. The activities of the round 4 variants onrebaudioside D at 0.0625 g/L SFP loading are listed in Table 8.3.

TABLE 8.3 Round 4 Variant SFP Activity on Rebaudioside D SEQ ID NO:Amino Acid Differences Increased (nt/aa) (Relative to SEQ ID NO: 232)RebM^(a) 349/350 L106T/L109R/R139P/G347D/E417A/Y421V/ + R427A 407/408L109R/R139P/S179L/E417R/Y421V/R427A − 355/356L106S/L109R/E112P/S131P/Q159R/G204D/ − G347D/E417A/Y421V/R427L 353/354F64P/L109R/E112P/R139P/E417A + 347/348L109R/S131P/R139P/S179V/R261P/Y396R/ +++ Y421V 363/364L106S/L109R/E112P/S131P/Q159R/S179L/ − E417A/Y421V ^(a)Levels ofincreased production were measured as μmol RebM/mg SFP-min at 0.0625 g/LSFP and 30 min and defined relative to SEQ ID NO: 232, as follows: “+” =production at least that of the reference polypeptide, but less than2-fold; “++” = at least 2-fold, but less than 3-fold; “+++” = at least3-fold, but less than 4-fold; and “++++” = at least 4-fold increasedproduction, as compared to the reference polypeptide.

Example 9 ADP-Glycosyltransferase Variants of SEQ ID NO: 348

In this Example, experiments for evolution and screening of GTpolypeptides derived from SEQ ID NO: 348, for improved glucosylation ofsteviol glycosides using ADP-glucose are described. Directed evolutionof the GT encoded by SEQ ID NO:347 (i.e., SEQ ID NO:348) was carried outby constructing libraries in which mutations associated with improvedactivity in previous rounds were recombined and in which mutationsidentified from homologs in publically available databases wererecombined. These libraries were then plated, grown, and screened usingthe high-throughput (HTP) assay described below to provide a fifth round(“Round 5”) of engineered GT variant polypeptides, 6 from library 5.01(Table 9.1) and 18 from the remaining libraries in the round (Table9.2), with glucosyltransferase activity toward ADP-glucose and steviolglycosides.

HTP Assay for Glucose Transfer from ADP-Glucose to Steviol Glycosides

Assays were performed on 96-well plates of cleared E. coli culturelysates expressing SEQ ID NO: 348 variants. Lysis buffer volume wasincreased to 400 uL from the 250 uL used in previous rounds, and thelysate was diluted 10-fold. For round 5.01, assays were conducted with10 μL lysate in 100 μL reactions and with substrate loading of 1 mMstevioside (ChromaDex, >94% purity), from a 20 mM stock solution in 50%ethanol and co-substrate loading of 1 mM ADP-glucose (Sigma, >93%purity). The following reaction conditions were used: 50 mM Tris-HClbuffer, pH 7.5, 3 mM MgCl₂, 40° C. in a Thermotron® titre-plate shakerwith 300 RPM shaking for 1 h. The reactions were quenched by adding 10μl assay 90 μL acetonitrile with 0.2% formic acid and centrifuged 10 mat 4° C. The supernatants were diluted 1:10 in water and analyzed byRapidFire SPE-MS/MS (Agilent) as described in Table 9.3. For theremaining round 5 libraries, the lysate was diluted 4-fold instead of10-fold, 50 mM potassium phosphate buffer pH 7 was used instead ofTris-HCl, the temperature was 50° C., the reaction time was 2h, and theassay was performed with both stevioside and rebaudioside D (Chromadex,>93%).

The engineered polypeptides are listed in Table 9.1 and Table 9.2.Although the parent and variant constructs contain an N-terminalhistidine tag for affinity purification, the mutations were numberedrelative to the untagged reference sequence for clarity. Shake-flaskscale cultures were grown, lysed, and lyophilized to powder as describedin Example 1 for SEQ ID NO: 500.

TABLE 9.1 Round 5.01 Variants and RebA Levels SEQ ID NO: Amino AcidDifferences Increased (nt/aa) (Relative to SEQ ID NO: 348) RebA^(a)499/500 L106S/E112P/G204D/G347D/R396Y/ +++ E417R/R427A/Q431D 501/502G204D/G347D/R396Y/E417R/Q431D ++ 503/504 L106S/E112P/G204D/G347D/R396Y/++ E417R/R427A 505/506 E112P/G204D/G347D/R396Y/E417R/ + R427A/Q431D507/508 L106S/E112P/G204D/G347D/R396Y/E417R + 509/510E112P/G204D/G347D/R396Y/E417R/R427A + ^(a)Levels of increased productionwere determined relative to the reference polypeptide of SEQ ID NO: 348,and defined as follows: “+” = production at least 10-fold that of thereference polypeptide, but less than 20-fold; “++” = at least 20-fold,but less than 30-fold; and “+++” = at least 30-fold increasedproduction, as compared to the reference polypeptide.

TABLE 9.2 Additional Round 5 Variants and RebA and RebM Levels SEQ IDNO: Amino Acid Differences Increased Increased (nt/aa) (Relative to SEQID NO: 348) RebA^(a) RebM^(b) 511/512 N137G/D161L/V435R ++++ ++++513/514 K38R/A178V/W401L ++ ++++ 515/516 R74W/R102K/N137G/D161L/ ++ +H259S/K289S 517/518 I14V/L100F ++ ++++ 519/520 D290E/A351G/W401L ++ +++521/522 I28M/I44V/V365I/A407E ++ +++ 523/524 K185R/D290E/W401L/I422M ++++ 525/526 D98P/I233W ++ +++ 527/528 T54P/V413L ++ +++ 529/530R102K/D161L/T250A/V435E ++ ++ 531/532 I92L/S118A ++ ++ 533/534K38R/S118A/D290E/A351G/ ++ ++ D375P/W401L/I422M 535/536 N137G/D169G + ++537/538 K38R/D290E/A351G/W401L/I422M + + 539/540 V435Q/M438A + ++541/542 Q159M/D169S/R173G/D300Q/ + + Q424E/M438A 543/544S118A/S156A/A178V/D290E/ + ++ D375P/W401L/I422M 545/546A110G/K222R/T250R/H259P/V435G + + ^(a)Levels of increased RebAproduction were determined relative to the reference polypeptide of SEQID NO: 348, and defined as follows: “+” = production at least that ofthe reference polypeptide, but less than 3-fold; “++” = production atleast 3-fold, but less than 6-fold; “+++” = at least 6-fold, but lessthan 9-fold; and “++++” = at least 9-fold increased production, ascompared to the reference polypeptide. ^(b)Levels of increased RebMproduction were determined relative to the reference polypeptide of SEQID NO: 348, and defined as follows: “+” = production at least that ofthe reference polypeptide, but less than 15-fold; “++” = production atleast 15-fold, but less than 30-fold; “+++” = at least 30-fold, but lessthan 45-fold; and “++++” = at least 45-fold increased production, ascompared to the reference polypeptide.

TABLE 9.3 RapidFire SPE-MS/MS Conditions for Steviol GlycosideDetection. Agilent RapidFire Conditions Buffer A 0.1% formic acid inLC/MS grade water; 1.5 mL/min flow rate Buffer B 0.1% formic acid inLC/MS grade methanol; 0.5 mL/min flow rate Aqueous wash Water Organicwash Acetonitrile SPE cartridge Agilent RapidFire cartridge A (C4) RFstate 1 600 ms RF state 2 2500 ms RF state 3 0 RF state 4 5000 ms RFstate 5 1000 ms Agilent Jet Stream source parameters Drying gas 325° C.temperature Drying gas flow 7 L/min Nebulizer pressure 50 psi Sheath gas300° C. temperature Sheath gas flow 12 L/min Capillary voltage +4000 VNozzle voltage +500 V Agilent 6470 Triple Quadrupole MRM parametersCompound Q1 Q3 Dwell Fragmentor CE CAV Stevioside 827.4 665.3 50 150 505 RebA 989.5 827.5 50 300 60 5 RebD or RebI 1151.7 827.5 50 285 55 5RebM 1313.7 827.5 50 350 70 5Shake Flask Powder Characterization Assay and Analysis for GlucosylTransfer from ADP-Glucose to Stevioside or Rebaudioside D

A shake flask powder loading dose response experiment was performed tocharacterize activity of the engineered round 5 variant SEQ ID NO: 500relative to SEQ ID NO: 348 on stevioside and rebaudioside D. Levels of0.03-1 g/L shake flask powder (SFP) were added to a 100 μL totalreaction volume containing 50 mM Tris-HCl buffer, pH 7.5, 3 mM MgCl₂,2.5% v/v ethanol with 1 mM stevioside (Chromadex, >94% purity) orrebaudioside D (ChromaDex, >93% purity) and 1 mM ADP-glucose. Thereaction was performed at 40° C. in a thermocycler for 0.5 h, and thereaction was quenched with 50 μL acetonitrile with 0.2% formic acid andprecipitated by centrifugation. The supernatant was diluted 1:67 inwater and analyzed for steviol glycosides by LC-MS/MS as described inExample 1, Table 1.1. At 1 g/L loading, SEQ ID NO: 500produced >1.2-fold as much rebaudioside A from stevioside as SEQ ID NO:348, and >1.07-fold as much rebaudioside M from rebaudioside D.Subsequently, SEQ ID NO: 499 (i.e., 500) was re-cloned to omit theN-terminal histidine tag, expressed in high throughput, and the plateswere lysed in 400 μL. The resulting powder, containing the variant SEQID NO: 548, was assayed relative to the SEQ ID NO: 500 variant, with 2.5μL lysate added to a 100 μL total reaction volume containing 50 mMpotassium phosphate buffer, pH7, 3 mM MgCl₂, 2.5% v/v ethanol with 1 mMstevioside (Chromadex, >94% purity) or rebaudioside D (ChromaDex, >93%purity) and 1 mM ADP-glucose. The reaction was performed at 50° C. in aThermotron® shaking incubator at 300 RPM for 4 h, and the reaction wasquenched by adding 10 μL assay to 90 μL acetonitrile with 0.2% formicacid and precipitated by centrifugation. The supernatant was diluted1:10 in water and analyzed for steviol glycosides by RapidFire SPE-MS/MSas described in Table 9.3. Under these conditions, the SEQ ID NO: 548variant produced nearly 1.5-fold more rebaudioside A from stevioside asthe SEQ ID NO: 500 variant, and over 1.6-fold more rebaudioside M fromrebaudioside D.

Subsequently, shake flask powder was prepared containing the variant ofSEQ ID NO: 548, and it was assayed relative to the SEQ ID NO: 348variant. Levels of 0.25-10 g/L shake flask powder (SFP) were added to a100 μL total reaction volume containing 50 mM potassium phosphatebuffer, pH 7, 3 mM MgCl₂, 2 mM ADP-glucose, and 1 g/L rebaudioside A 60(60% rebaudioside A, >35% stevioside) or rebaudioside D (Chromadex, >93%purity). The reaction was performed at 40° C. in a thermocycler for 1 h,and the reaction was quenched by adding 10 μL assay to 90 μLacetonitrile with 0.2% formic acid and precipitated by centrifugation.The supernatant was diluted 1:10 in water and analyzed for steviolglycosides by LC-MS/MS as described in Example 1, Table 1.1. The SEQ IDNO: 548 variant reached >65% conversion of rebaudioside A 60 to amixture of rebaudioside A and rebaudioside I and nearly completeconversion of rebaudioside D to rebaudioside M. At 0.25 g/L shake flaskpowder loading, the SEQ ID NO: 548 variant reached 1.4-fold theconversion of rebaudioside D as the SEQ ID NO: 348 variant, and 2.2-foldthe conversion of rebaudioside A 60.

Expression Analysis of AGT Enzymes

The six rounds of engineered AGT polypeptides for β-1,3-glucosylation ofsteviol glycosides were analyzed by polyacrylamide gel-electrophoresisto determine relative protein expression levels. Samples were preparedwith 1×LDS loading buffer and 1× reducing agent (Life Technologies). A4-12% Bis-Tris acrylamide gel (Life Technologies) was loaded with 5 μgper lane of lyophilized soluble crude lysate from shake flask scalecultures and run with MES running buffer for 25 min at 200 V, and bandswere quantified using Image J analysis software. The relative expressionlevels are listed in Table 9.4. SEQ ID NOs: 547/548, 499/500, and347/348 are significantly better expressed genes and/or significantlybetter folded/more stable proteins. Thus, these genes produced moreprotein than the wild-type gene.

TABLE 9.4 Protein Levels of Engineered AGT Variants SEQ ID NO: IncreasedProtein^(a) 548 +++ 500 ++ 348 ++ 232 + 32 − 8 − ^(a)Levels of increasedprotein were determined relative to the reference polypeptide of SEQ IDNO: 4 and defined as follows: “+” = band intensity at least that of thereference polypeptide, but less than 2-fold; “++” = band intensity atleast 2-fold, but less than 4-fold; and “+++” = at least 4-foldincreased band intensity, relative to the reference polypeptide.

Example 10 Transformation of Rebaudioside A to Rebaudioside I with AGT(SEQ ID NO: 500)

In this Example, scale-up of the reaction transforming rebaudioside A torebaudioside I using the SEQ ID NO: 500 variant, and the isolation andcharacterization of rebaudioside I are described. A reaction containing5 g/L of the SEQ ID NO: 500 variant lyophilized shake flask powder, 50mM potassium phosphate buffer pH 7, 10.3 mM magnesium chloride, 10 g/Lrebaudioside A (>97% purity), and 10.3 mM ADP-glucose (Sigma) with 10 mLtotal volume was stirred on a stir plate at 300 RPM and 35° C. for 89 h.The reaction was transparent when it was begun, and by the end of thereaction it was a white emulsion. The assay was diluted 1:10 in water,and 10 μL of diluted assay was added to 90 μL acetonitrile with 0.2%formic acid and precipitated by centrifugation. The supernatant wasdiluted 1:10 in water and analyzed for steviol glycosides by LC-MS/MS asdescribed in Example 1, Table 1.1. This analysis confirmed production ofrebaudioside I at high conversion. Because of its low solubility,rebaudioside I settled. Therefore, the isolation was conducted byremoving the supernatant, resuspending the sediment with the minimalvolume of water, and centrifuging. This wash step was repeated twice,and a brown sediment at the surface of the white sediment was scrapedoff. The material was lyophilized and analyzed by Acorn NMR. Thematerial was dissolved in pyridine-d5, and ¹H 1-D, ¹³C DEPT-135, ¹H-¹³CHSQC NMR experiments were performed with a Varian Inova 500 NMRspectrometer. The NMR spectra were completely consistent with literaturereports for rebaudioside I (see Table 10.1). See FIG. 3 for thestructure of rebaudioside I with carbons numbered.

TABLE 10.1 NMR Characterization of Rebaudioside I Position δ ¹³C,ppm^(a) δ ¹H, ppm ¹H Multiplicity (J,Hz) 1  40.4 0.73 t(13.4) 1  40.41.74 m 2  19.1 1.42 m 2  19.1 2.18 m 3  38.2 1.02 M 3  38.2 2.35 m 4  —— 5  57 1.03 m 6  21.9 1.88 m 6  21.9 2.32 m 7  41.5 1.29 m 7  41.5 1.31m 8  — — 9  53.9 0.88 d(7.2) 10  — — 11  20.3 1.69 m 11  20.3 1.7 m 12 37 1.97 m 12  37 2.27 m 13  — — 14  44.1 1.79 m 14  44.1 2.59  d(11.9)15  47.4 2.05 Brs 16  — — 17  104.5 5.02 s 17  104.5 5.66 s 18  28.11.22 s 19  — — 20  15.4 1.27 s 1′  95 6.14 d(8.2) 2′  72.3 4.17 m 3′ 89.4 4.25 m 4′  69 4.23 m 5′  77.8 3.91 m 6′  61.4 4.33 m 6′  61.4 4.25m 1″   97.8 5.05 d(7.8) 2″   80.4 4.32 m 3″   87.5 4.17 m 4″   69.9 3.95m 5″   77.3 3.79 m 6″   62.3 4.16 m 6″   62.3 4.49 m 1′′′  104.4 5.55d(7.7) 2′′′  76 4.18 m 3′′′  78.3 4.26 or 4.23 m 4′′′  71.9 4.22 m 5′′′ 78.5 3.92 m 6′′′  62.9 4.39 m 6′′′  62.9 4.51 m 1′′′′ 104.5 5.35 d(7.9)2′′′′ 75.1 or 75.3 4.03 m 3′′′′ 78.3 4.26 m 4′′′′ 71.2 or 71.4 4.12 m5′′′′ 78.3 4.11 m 6′′′′ 62.1 4.24 m 6′′′′ 62.1 4.56 m 1′′′′′ 104.8 5.26d(7.9) 2′′′′′ 75.1 or 75.3 4.03 M 3′′′′′ 78.3 4.26 or 4.23 M 4′′′′′ 71.2or 71.4 4.12 M 5′′′′′ 78.3 4 M 6′′′′′ 62.2 4.24 M 6′′′′′ 62.2 4.54 M^(a)Dried material was characterized by ¹H, DEPT-135, and HSQC inpyridine-d5 on a Varian Inova 500 NMR instrument. The spectra werecompared to literature spectra (See e.g., Prakash et al., Molecules,19(11): 17345-55 [2014]) in order to assign peaks. ¹³C signals are fromDEPT-135. Some peaks were too close in shift to determine assignment;when this occurred both options are listed.

Example 11 ADP-Glycosyltransferase Variants of SEQ ID NO: 548

In this Example, experiments for evolution and screening of GTpolypeptides derived from SEQ ID NO: 548 for improved glucosylation ofsteviol glycosides using ADP-glucose are described. Directed evolutionof the GT encoded by SEQ ID NO:547 (i.e., SEQ ID NO:548) was carried outby constructing libraries in which mutations associated with improvedactivity in previous rounds were recombined. This library was thenplated, grown, and screened using the high-throughput (HTP) assaydescribed below to provide a sixth round (“Round 6”) of 66 engineered GTvariant polypeptides with glucosyltransferase activity towardADP-glucose and steviol glycosides (Table 11.1).

HTP Assay for Glucose Transfer from ADP-Glucose to Steviol Glycosides

Assays were performed on 96-well plates of cleared E. coli culturelysates expressing SEQ ID NO: 548 variants. Lysis buffer volume was 400uL, and the lysate was diluted 4-fold. The assay was conducted with 10μL lysate in 100 μL reactions and with substrate loading of 1 mMstevioside (ChromaDex, >94% purity), from a 20 mM stock solution in 50%ethanol and co-substrate loading of 1 mM ADP-glucose (Sigma, >93%purity). The following reaction conditions were used: 50 mM potassiumphosphate buffer, pH 7, 3 mM MgCl₂, 50° C. in a Thermotron® titre-plateshaker with 300 RPM shaking for 4 h. The reactions were quenched byadding 10 μl assay 90 μL acetonitrile with 0.2% formic acid andcentrifuged 10 m at 4° C. The supernatants were diluted 1:10 in waterand analyzed by RapidFire SPE-MS/MS as described in Table 9.3. The top84 variants were retested with the same conditions using 1 mM stevioside(Chromadex, >94% purity), 1 mM rebaudioside A (>97% purity), orrebaudioside D (Chromadex, >93% purity). The resulting engineered GTvariant polypeptides are listed in Table 11.1. Shake-flask scalecultures were grown, lysed, and lyophilized to powder as described inExample 1 for variants with SEQ ID NOS: 554, 562, 568, and 576.

TABLE 11.1 Round 6 Variants and RebA, RebI, and RebM Levels SEQ ID NO:Amino Acid Differences Increased Increased Increased (nt/aa) (Relativeto SEQ ID NO: 548) RebA^(a) RebI^(a) RebM^(a) 549/550I28M/R74W/D161L/D290E/V365I +++ +++ + 551/552I28M/I44V/I92L/D161L/K222R/D300Q/V413L +++ + + 553/554I28M/R74W/S156A/D161L/V365I/A407E +++ ++ + 555/556I28M/R74W/S156A/Q159M/D161L/A178V/ +++ + + D300Q/V365I/V435Q/M438A557/558 I14V/I28M/I92L/L100F/R102K/Q159M/ +++ + +D161L/I233W/A351G/I422M 559/560 I14V/R74W/D161L/D375P/W401L +++ − +561/562 I14V/I28M/K38R/R74W/L100F/R102K/S118A/D161L/ +++ + +D169G/A178V/I233W/T250R/A407E/I422M/M438A 563/564I28M/K38R/I92L/D98P/L100F/R102K/S156A/D161L +++ + + 565/566I14V/I28M/I92L/L100F/R102K/Q159M/ +++ − + D161L/D169S/I233W 567/568I28M/I44V/S118A/S156A/D161L/K222R/ +++ + − K289S/V435Q/M438A 569/570I28M/I92L/L100F/R102K/A110G/D161L/ +++ − + K185R/T250A/D300Q/D375P/V435Q571/572 I14V/I28M/I44V/D161L/D169S +++ + + 573/574I28M/S156A/D161L/I233W/H259S/D300Q/V435R +++ ++ + 575/576I14V/I28M/T54P/D161L/K185R/V413L +++ ++ + 577/578I14V/I28M/K38R/L100F/R102K/A110G/ ++ + + Q159M/D161L/I233W/H259S/D290E/D300Q/A351G/V435Q 579/580 I28M/S156A/D161L/K185R/V435R/M438A ++ ++ +581/582 I28M/I92L/D98P/L100F/A110G/S156A/D161L/W401L ++ − − 583/584I28M/D98P/L100F/R102K/D161L/K185R/ ++ − + A351G/W401L/V435E/M438A585/586 I14V/I28M/L100F/N137G/S156A/D161L/ ++ + +K222R/H259S/K289S/V365I/W401L/V435R/M438A 587/588I14V/I28M/I92L/L100F/D161L/K222R/I233W/ ++ − + K289S/D300Q 589/590R102K/N137G/Q159M/D161L/I422M/Q424E ++ − + 591/592I14V/I28M/L100F/S156A/D161L/T250R ++ + + 593/594I14V/I28M/R74W/D98P/L100F/R102K/ ++ − −D161L/R173G/A178V/I233W/T250A/H259S/ D290E/A407E 595/596I14V/I28M/K38R/A110G/N137G/D161L/ ++ − − K222R/K289S/W401L 597/598I28M/T54P/I92L/Q159M/D161L/D290E ++ + + 599/600I14V/I28M/I44V/R74W/D98P/R102K/ ++ − −N137G/Q159M/D161L/K185R/K222R/T250A 601/602I14V/I28M/R102K/S118A/N137G/D161L/ ++ − + K185R/K222R/T250R/H259P/W401L603/604 Q159M/D161L/K222R/D290E/D375P/A407E ++ − + 605/606I14V/A110G/S156A/D161L/D375P/W401L/ ++ − + V435E/M438A 607/608I14V/I92L/D98P/L100F/Q159M/D161L/ ++ − + H259P/V365I/I422M/Q424E/V435R609/610 K38R/D161L/D300Q/M438A ++ − + 611/612R74W/S156A/D161L/R173G/A178V/Q424E/V435E ++ − − 613/614R74W/D98P/L100F/R102K/S118A/S156A/ ++ − − Q159M/D161L/V435E/M438A615/616 I14V/Q159M/D161L/V365I/V435E/M438A ++ − − 617/618N137G/Q159M/D161L/K185R/D300Q/ ++ − − A351G/V365I/V435Q 619/620I14V/R74W/A110G/Q159M/D161L/D169G/ ++ − −R173G/T250A/H259P/D290E/D375P/A407E/I422M 621/622I14V/D161L/K222R/T250R/H259S/K289S/ ++ − − D375P/W401L/V413L 623/624Q159M/D161L/W401L ++ − − 625/626 I44V/R74W/L100F/R102K/D161L/I233W/ ++ −− V365I/V435G/M438A 627/628 I14V/D161L/D300Q + − − 629/630I28M/I44V/R74W/W401L + + − 631/632 I14V/D161L/K222R/T250R/V435Q/M438A +− − 633/634 R74W/D98P/L100F/R102K/A110G/S118A/ + − −D161L/A178V/T250R/K289S/D290E/ D300Q/V435E/M438A 635/636S156A/Q159M/D161L/D169S + − − 637/638I14V/K38R/L100F/R102K/D161L/R173G/ + − −A178V/K222R/T250R/D375P/W401L/V413L 639/640D98P/L100F/S156A/Q159M/D161L/A178V/ + − − H259S/K289S/D290E/A351G/I422M641/642 I14V/K38R/R74W/A110G/S156A/D161L/ + − − R173G/A178V/K222R/D300Q643/644 D98P/L100F/S118A/Q159M/D161L/D300Q + − − 645/646I14V/I28M/I92L/L100F/R102K/I422M/Q424E/V435Q + +++ + 647/648I14V/I28M/Q159M/V365I/V435Q + +++ + 649/650I28M/I92L/S118A/Q159M/K222R/T250R/ + + + H259P/D300Q/A407E 651/652D161L/H259S/K289S/V435R/M438A + − − 653/654 I28M + ++ + 655/656I28M/T54P/Q159M/D290E/M438A + ++ + 657/658I14V/I28M/Q159M/K289S/D290E/D300Q + ++ + 659/660I28M/I44V/K289S/D290E/A351G/I422M + +++ + 661/662I28M/I44V/V435R/M438A + ++ + 663/664 I14V/I28M/I44V/I92L + ++ + 665/666I14V/I28M/A351G + ++ + 667/668 I28M/I44V/D98P/L100F/R102K/S118A + ++ +669/670 I14V/I28M/I44V/D375P + + + 671/672 I14V/I28M/T54P/V365I + +++ +673/674 I14V/I28M/K38R/R74W/R102K/S156A/Q159M/ + ++ +I233W/T250A/K289S/V413L/I422M/Q424E/V435R/ M438A 675/676R74W/D375P/V435G + − − 677/678 S118A + + − 679/680 I28M/T54P/T250R/K439N− ++ + ^(a)Levels of increased production were determined relative tothe reference polypeptide of SEQ ID NO: 548 and defined as follows: “−”= production less than that of the reference polypeptide; “+” =production at least that of the reference polypeptide, but less than1.5-fold; “++” = at least 1.5-fold, but less than 2-fold; and “+++” = atleast 2-fold increased production, as compared to the referencepolypeptide.Shake Flask Powder Characterization Assay and Analysis for GlucosylTransfer from ADP-Glucose to Stevioside or Rebaudioside D

A shake flask powder loading dose response experiment was performed tocharacterize activity of the engineered round 6 variants on steviosideand rebaudioside D. Levels of 0.03-1 g/L shake flask powder (SFP) wereadded to a 100 μL total reaction volume containing 50 mM potassiumphosphate buffer, pH 7, 3 mM MgCl₂, 2.5% v/v ethanol with 1 mMstevioside (Chromadex, >94% purity) or rebaudioside D (ChromaDex, >93%purity) and 1 mM ADP-glucose. The reaction was performed at 50° C. in aThermotron® titre-plate shaker at 300 RPM for 1h, and the reaction wasdiluted with 150 μL water and then quenched by transferring 12.5 μL into87.5 μL acetonitrile with 0.2% formic acid and precipitated bycentrifugation. The supernatant was diluted 1:20 in water and analyzedfor steviol glycosides by RapidFire SPE-MS/MS as described in Example 9,Table 9.3. The relative productivities of the enzymes are listed inTable 11.2.

TABLE 11.2 Round 6 Variant SFP Activity on Stevioside and Rebaudioside DSEQ ID NO: Amino Acid Differences Increased Increased (nt/aa) (Relativeto SEQ ID NO: 548) RebA^(a) RebM^(a) 576I14V/I28M/T54P/D161L/K185R/V413L +++ ++ 568I28M/I44V/S118A/S156A/D161L/K222R/K289S/V435Q/M438A +++ ++ 554I28M/R74W/S156A/D161L/V365I/A407E ++++ + 562I14V/I28M/K38R/R74W/L100F/R102K/S118A/D161L/D169G/ ++ ++A178V/I233W/T250R/A407E/I422M/M438A ^(a)Levels of increased activitywere measured as fold-change relative to SEQ ID NO: 548 in μmol RebA orRebM/mg SFP-min at 0.0625 g/L SFP and 60 mm and defined as follows: “+”= activity at least that of the reference polypeptide, but less than2-fold; “++” = at least 2-fold, but less than 3-fold; “+++” = at least3-fold, but less than 4-fold; and “++++” = at least 4-fold increasedactivity, as compared to the reference polypeptide.

Example 12 ADP-Glycosyltransferase Variants of SEQ ID NO: 562

In this Example, experiments for evolution and screening of GTpolypeptides derived from SEQ ID NO: 562 for improved glucosylation ofsteviol glycosides using ADP-glucose are described. Directed evolutionof the GT encoded by SEQ ID NO:561 (i.e., SEQ ID NO:562) was carried outby constructing libraries in which mutations associated with improvedactivity in previous rounds were recombined. This library was thenplated, grown, and screened using the high-throughput (HTP) assaydescribed below to provide a seventh round (“Round 7”) of 37 engineeredGT variant polypeptides with glucosyltransferase activity towardADP-glucose and steviol glycosides (Table 12.1).

HTP Assay for Glucose Transfer from ADP-Glucose to Steviol Glycosides

Assays were performed on 96-well plates of cleared E. coli culturelysates expressing SEQ ID NO: 562 variants. Lysis buffer volume was 400uL, and the lysate was diluted 4-fold. The assay was conducted with 10μL lysate in 100 μL reactions and with substrate loading of 1 mMstevioside (ChromaDex, >94% purity), from a 20 mM stock solution in 50%ethanol and co-substrate loading of 1 mM ADP-glucose (Sigma, >93%purity). The following reaction conditions were used: 50 mM potassiumphosphate buffer, pH 7, 3 mM MgCl₂, 50° C. in a Thermotron® titre-plateshaker with 300 RPM shaking for 1 h. The reactions were quenched byadding 10 μl assay 90 μL acetonitrile with 0.2% formic acid andcentrifuged 10 m at 4° C. The supernatants were diluted 1:10 in waterand analyzed by RapidFire SPE-MS/MS as described in Table 9.3. The top84 variants were retested with the same conditions using 1 mM stevioside(Chromadex, >94% purity), 1 mM rebaudioside A (>97% purity), orrebaudioside D (Chromadex, >93% purity). The resulting engineered GTvariant polypeptides are listed in Table 12.1.

TABLE 12.1 Round 7 Variants and RebA, RebI, and RebM Levels SEQ ID NO:Amino Acid Differences Increased Increased Increased (nt/aa) (Relativeto SEQ ID NO: 562) RebA^(a) RebI^(b) RebM^(b) 681/682I75L/M87W/A110G/N137G/G169S/S191R/ +++ − − A199K/K208A/Q209G/K289S/V435Q683/684 M87W/G169S/S191R/A199K/I207L/Q209G/ +++ − − W401L/V413L 685/686I44V/M87W/N137G/Q159M/G169S/S191R/ +++ − − A199K/K208D 687/688I44V/M87W/A199K/K208A +++ − − 689/690 M87W/I92L/Q159M/S191R/A199K/K208A/+++ − − Q209G/K289S/D290E 691/692N137G/I198M/A199K/I207L/K208D/A426V/V435R ++ ++ + 693/694I75L/S76R/M87W/S191R/Q197R/A199K/ ++ − − Q209G/D300Q 695/696I75L/S76R/M87W/S191R ++ ++ + 697/698V19L/I75L/S76R/M87W/I92L/A199K/I207L/K208A ++ − − 699/700N137G/A199K/K208A/Q209G/D290E/V435R ++ − − 701/702G169S/Q197R/A199K/I207L/Q209G/K222R/ ++ − − D300Q/V413L/V435R 703/704G169S/S191R/Q197R/A199K/I207L/K208D ++ − − 705/706I75L/A110G/Q197R/A199K/K208A/D290E/ ++ − − D300Q/W401L/V413L 707/708I92L/N137G/S191R/A199K/Q209G ++ − − 709/710M87W/I92L/A110G/G169S/A199K/I207L/ ++ − − Q209G/D290E/D300Q 711/712S76R/I92L/A199K/Q209G + − − 713/714 M87W/N137G/V435R + ++++ + 715/716I44V/S76R/Q197R/A199K/K208D/A351G + − − 717/718 I75L/M87W/D300Q + +++ +719/720 V19L/I44V/A110G/S191R/I198M/A199K/ + − + K208A/D300Q/V365I721/722 I44V/M87W/I92L/N137G/Q159M/A199K/Q209G + − − 723/724I75L/S76R/M87W/I92L/D290E/D300Q + ++ − 725/726M87W/S191R/I198M/A199K/K222R/P244L/ + − − K289S/D300Q/V435R 727/728M87W/I92L/K208A/W401L + + − 729/730 Q197R/I198M/A199K/K208D/Q209G + − −731/732 M87W/I92L/Q159M/G169S/S191R/I198M/ + − − D290E/V413L/V435Q733/734 S191R/I207L/K208A/K289S/D290E/V413L/V435Q + + ++ 735/736N137G/V365I + ++ + 737/738 V19L/M87W/I92L/S191R/A199K/Q209G/ + − −V413L/V435Q 739/740 I75L/S76R/M87W/I92L + − − 741/742I75L/M87W/I92L/G169S/I207L/K208A/ + + − D300Q/V413L/V435Q 743/744I44V/M87W/N137G/S191R/Q197R/Q209G/ + − − K289S/W401L 745/746I92L/Q197R/A199K/I207L/K208D/W401L + − − 747/748I75L/M87W/A110G/S191R/Q197R/I198M/I207L/ + − −K208D/K289S/D290E/D300Q/W401L/V413L 749/750Q159M/Q197R/A199K/I207L/Q209G + − − 751/752M87L/I92L/Q197R/I198M/A199K/K208D/D300Q + − − 753/754 M87W/V435R + − −^(a)Levels of increased production were determined relative to thereference polypeptide of SEQ ID NO: 562, and defined as follows: “−” =production less than that of the reference polypeptide; “+” = productionat least that of the reference polypeptide, but less than 3-fold; “++” =at least 3-fold, but less than 5-fold; and “+++” = at least 5-foldincreased production, as compared to the reference polypeptide.^(b)Levels of increased production were determined relative to thereference polypeptide of SEQ ID NO: 562, and defined as follows: “−” =production less than that of the reference polypeptide; “+” = productionat least that of the reference polypeptide, but less than 1.5-fold; “++”= at least 1.5-fold, but less than 2-fold; “+++” = at least 2-fold, butless than 2.5-fold; and “++++” = at least 2.5-fold increased production,as compared to the reference polypeptide.

Example 13 Synthesis, Optimization, and Assaying of GlycosyltransferaseEnzymes with Glucosylation Activity

In this Example, methods used in the synthesis, optimization andassaying of UGT enzymes with glucosylation activity are described.

Gene Synthesis and Optimization

Polynucleotide sequences encoding glycosyltransferases from Oryza sativaJaponica, Solanum tuberosum, Lycium barbarum, and Solanum lycopersicumwere codon-optimized and synthesized as the genes of SEQ ID NOS: 755,757, 759, 761, 763, 765, and 767. These synthetic genes were cloned intoa pCK110900 vector system (See e.g., US Pat. Appln. Publn. No.2006/0195947, which is hereby incorporated by reference) andsubsequently expressed in E. coli W3110 (ΔfhuA). The E. coli strainW3110 expressed the UGT enzymes under the control of the lac promoter.

Production of Shake Flask Powders (SFP)

A shake-flask procedure was used to generate the glycosyltransferasepolypeptide shake flask powders (SFP) for characterization assays usedin the biocatalytic processes described herein. Shake flask powder (SFP)preparation of enzymes provides a more purified preparation (e.g., upto >30% of total protein) of the enzyme as compared to the cell lysateused in HTP assays and also allows for the use of more concentratedenzyme solutions. A single colony of E. coli containing a plasmidencoding an engineered polypeptide of interest was inoculated into 5 mLLuria Bertani broth containing 30 μg/ml chloramphenicol and 1% glucose.Cells were grown overnight (at least 16 hours) in an incubator at 30° C.with shaking at 250 rpm. The culture was diluted into 250 mL TerrificBroth (12 g/L bacto-tryptone, 24 g/L yeast extract, 4 mL/L glycerol, 65mM potassium phosphate, pH 7.0, 1 mM MgSO₄) containing 30 μg/ml CAM, ina 1 L flask to an optical density of 600 nm (OD600) of 0.2 and allowedto grow at 30° C.

Expression of the glycosyltransferase gene was induced by addition ofIPTG to a final concentration of 1 mM when the OD600 of the culture was0.6 to 0.8. Incubation was then continued overnight (at least 16 hours).Cells were harvested by centrifugation (5000 rpm, 15 min, 4° C.) and thesupernatant discarded. The cell pellet was resuspended in two volumes of25 mM triethanolamine buffer, pH 7.5, and passed through aMICROFLUIDIZER® high pressure homogenizer (Microfluidics), with standardE. coli lysis settings and maintained at 4° C. Cell debris was removedby centrifugation (10,000 rpm, 45 minutes, 4° C.). The cleared lysatesupernatant was collected and frozen at −80° C. and then eitherHis-affinity purified and dialyzed to produce purified protein orlyophilized to produce a dry shake-flask powder of crude protein.

Assay for Rebaudioside A Glucosylation with Purified Proteins

First, 50 μL purified protein was diluted in 200 μL total reactionvolume consisting of 50 mM Tris-HCl buffer pH 7.5, 3 mM magnesiumchloride, 1 mM rebaudioside A, and 0.5 mM uridine diphosphoglucose. Thereaction was performed at 30° C. in a Thermotron® titre-plate shakerwith 300 RPM shaking for 18 h. Boiled enzyme reaction was used as thenegative control. Ten L of the reaction was quenched with 90 μLacetonitrile with 0.2% formic acid and precipitated by centrifugation.Glycosylated rebaudioside A products were detected in the supernatant byLC-MS/MS as described in Example 1, Table 1.1.

Production of rebaudioside D from rebaudioside A was detected for SEQ IDNO: 756, 758, 762, and 768. Some enzymes also produced a regioisomer ofrebaudioside D, likely the 6-linked molecule rebaudioside D2. Despitepoor soluble expression, SEQ ID NO: 758 demonstrated high specificactivity and good selectivity toward producing β-1,2-glucose linkages inthe steviol glycoside substrates.

Assay for Rebaudioside A Glucosylation with Shake Flask Powder

Lyophilized shake flask powder was reconstituted to 20 mg/mL. Then, 10μL of these stocks were diluted in 100 μL total reaction volume of 50 mMpotassium phosphate (KPhos) buffer, pH 7, with 3 mM MgCl₂, 1 mMrebaudioside A (>97% purity), and 2 mM uridine diphosphoglucose(UDP-glucose). The reaction was performed at 40° C. in a Thermotron®titre-plate shaker with 300 RPM shaking for 16-18 h. Activity wasdetected for SEQ ID NO: 758 over negative control. Low conversion (i.e.,<10%), of rebaudioside A to rebaudioside D was observed in the LC-MS/MSanalysis.

Example 14 GT Variants of SEQ ID NO: 758

In this Example, experiments for evolution and screening of GTpolypeptides derived from SEQ ID NO: 758 for improved glucosylation ofsteviol glycosides are described. Directed evolution of the GT encodedby SEQ ID NO: 757 (i.e., SEQ ID NO: 758) was carried out by constructingcombinatorial libraries of variant genes in which positions associatedwith surface residues of the enzyme were subjected to mutagenesis. Theselibraries were then plated, grown, and screened using thehigh-throughput (HTP) assay described below to provide a first round(“Round 1”) of 10 engineered GT variant polypeptides withβ-1,2-glucosyltransferase activity toward steviol glycosides.

HTP Growth, Expression, and Lysate Preparation

Cells were picked into 96-well plates and grown overnight in LB mediacontaining 1% glucose and 30 μg/mL CAM, 30° C., 200 rpm, 85% humidity.Then, 20 μL of overnight growth were transferred to a deep-well platecontaining 380 μL TB growth media containing 30 μg/mL CAM, induced with1 mM IPTG, and incubated for 18 h at 30° C., 200 rpm, 85% humidity. Cellcultures were centrifuged at 4000 rpm, 4° C. for 10 m, and the mediadiscarded. Cell pellets thus obtained were frozen at −80° C., and lysedin 250 μL lysis buffer (0.5 g/L lysozyme and 0.5 g/L PMBS in 20 mMTris-HCl buffer, pH 7.5) with low-speed shaking for 2 h on titre-plateshaker at room temperature. The plates were then centrifuged at 4000 rpmand 4° C. for 20 min and the cleared lysate supernatants were used inthe HTP assay reactions described below.

HTP Assay for Rebaudioside A Glucosylation

Assays were performed on 96-well plates of cleared E. coli culturelysates expressing SEQ ID NO: 757 variants with lysate loading of 25 μLlysate in 100 μL reactions and with substrate loading of 1 mMrebaudioside A (Sigma, >96% purity), from a 20 mM stock solution in 50%ethanol, and co-substrate loading of 0.5 mM UDP-glucose (Sigma, >98%purity). The following reaction conditions were used: 50 mM Tris-HClbuffer, pH 7.5, 3 mM MgCl₂, 30° C. in a Thermotron® titre-plate shakerwith 300 RPM shaking for 4 h. The reactions were quenched with 0.5volume/volume acetonitrile with 0.2% formic acid and precipitated bycentrifugation for 10 m at 4° C. Glycosylated products were detected inthe supernatant by LC-MS/MS following 1:20 dilution in water with theinstrument and parameters described in Example 1, Table 1.1.Glycosyltransferase variant polypeptides that produced rebaudioside Dfrom rebaudioside A at greater quantities than SEQ ID NO: 758 wereidentified. The engineered polypeptides are listed in Table 14.1.Shake-flask scale cultures were grown for lyophilized powder productionas described in Example 1 for analysis of variants with the followingamino acid mutations shown in Table 14.1, relative to SEQ ID NO: 758.

TABLE 14.1 Round 1 Variants and RebD Levels SEQ ID NO: Amino AcidDifferences Increased (nt/aa) (Relative to SEQ ID NO: 758) RebD^(a)769/770 R69H/Y173N/V243A/M383V/T399A +++ 771/772Y173H/P175S/N191D/M365I/M383V/T399A +++ 773/774I56T/N191D/L354I/M383V/T399A +++ 775/776 R69H/Y173N/P175S/V243A/M246K/+++ L354I/M365I/M383V/T399A 777/778 F70L/N225G/I413V ++ 779/780F70L/N225G/E247G ++ 781/782 F70L/N225G/M246P/E409K/I413V ++ 783/784F70L/Q115S/N225G/E409K ++ 785/786 H74T/K310D/G396E/N424S ++ 787/788H74T/G396E + ^(a)Levels of increased production were determined relativeto the reference polypeptide of SEQ ID NO: 4, and defined as follows:“−” = production less than the reference polypeptide; “+” = productionat least that of the reference polypeptide, but less than 3-fold; “++” =at least 3-fold, but less than 5-fold; and “+++” = at least 5-foldincreased production, as compared to the reference polypeptide.

Shake Flask Lysate Characterization Assay and Analysis for RebaudiosideGlucosylation

First, 250 mL shake flask cultures were grown, induced, and lysed. Celldebris was removed by centrifugation as described in Example 1, and thecleared lysate supernatant was collected. Then, 10 μL of the lysate werediluted in 100 μL total reaction volume of 50 mM Tris-HCl buffer, pH7.5, 3 mM MgCl₂, 1 mM rebaudioside A (Sigma, >96% purity), and 2 mMUDP-glucose (Sigma>98% purity). The reaction was performed at 30° C. ina Thermotron® titre-plate shaker with 300 RPM shaking for 0-18 h. Thereaction described above was quenched with 0.5 volume/volumeacetonitrile with 0.2% formic acid and precipitated by centrifugation.Glycosylated products were detected in the supernatant by LC-MS/MSfollowing 1:20 dilution in water with the instrument and parametersdescribed in Example 1, Table 1.1.

TABLE 14.2 Shake-flask Characterization of Round 1 Variants and RebDLevels SEQ ID NO: Amino Acid Differences (nt/aa) (Relative to SEQ ID NO:758) Increased RebD^(a) 769/770 R69H/Y173N/V243A/M383V/T399A + 771/772Y173H/P175S/N191D/M365I/ + M383V/T399A 777/778 F70L/N225G/I413V −779/780 F70L/N225G/E247G − 785/786 H74T/K310D/G396E/N424S − 787/788H74T/G396E − ^(a)Levels of increased production were determined relativeto the reference polypeptide of SEQ ID NO: 758, at 4 h and defined asfollows: “−” = production less than the reference polypeptide; and “+” =production at least that of the reference polypeptide, but less than1.5-fold increased production, as compared to the reference polypeptide.

Variants corresponding to SEQ ID NOS:770 and 772 produced rebaudioside Dfrom rebaudioside A at greater quantities than the variant of SEQ ID NO:758. The variant of SEQ ID NO: 770 exhibited the highest activity onRebA. Thus, the encoding polynucleotide (SEQ ID NO: 769) was selectedfor further directed evolution.

Example 15 ADP-Glycosyltransferase Variants of SEQ ID NO: 770

In this Example, experiments for evolution and screening of GTpolypeptides derived from the SEQ ID NO: 770 variant for improvedglucosylation of steviol glycosides using ADP-glucose are described.Directed evolution of the GT encoded by SEQ ID NO: 769 (i.e., SEQ ID NO:770) was carried out by constructing libraries of variant genes.Libraries recombined beneficial mutations identified in Example 14(Round 1), combinatorially incorporated diversity from homologs inpublically available databases, or subjected certain structural featuresof the enzyme with saturation mutagenesis. These libraries were thenplated, grown, and screened using the high-throughput (HTP) assaydescribed below to provide a second round (“Round 2”) of engineered GTvariant polypeptides with glucosyltransferase activity towardADP-glucose and steviol glycosides. 10 engineered variants wereidentified from the recombined beneficial mutations (Table 15.1), 19from saturation mutagenesis (Table 15.2), and 53 from surface residueand homolog diversity (Table 15.3).

HTP Assay for Glucose Transfer from ADP-Glucose to Rebaudioside A

Assays were performed on 96-well plates of cleared E. coli culturelysates expressing SEQ ID NO: 769 variants with lysate loading of 25 μLlysate in 100 μL reactions and with substrate loading of 1 mMrebaudioside A (Sigma, >96% purity) and co-substrate loading of 4 mMADP-glucose (Sigma, >93% purity). The following reaction conditions wereused: 50 mM Tris-HCl buffer, pH 7.5, 3 mM MgCl₂, 30° C. in a Thermotron®titre-plate shaker with 300 RPM shaking for 18 h. The reactions werequenched with 0.5 volume/volume acetonitrile with 0.2% formic acid andprecipitated by centrifugation for 10 m at 4° C. Glycosylated productswere detected in the supernatant by LC-MS/MS following 1:10 dilution inwater with the instrument and parameters described in Example 1, Table1.1. Glycosyltransferase variant polypeptides that produced rebaudiosideD from rebaudioside A with ADP-glucose, at greater quantities than SEQID NO: 770 were identified. The engineered polypeptides from therecombined beneficial mutations are listed in Table 15.1. The engineeredpolypeptides from the saturation mutagenesis libraries are listed inTable 15.2.

TABLE 15.1 Combinatorial Round 2 AGT Variants and RebD Levels SEQ ID NO:Amino Acid Differences (nt/aa) (Relative to SEQ ID NO: 770) IncreasedRebD^(a) 789/790 Y24E/S28N/F262Y/C264S ++++ 791/792Y24E/S28N/F262Y/C264S/K423E +++ 793/794 S28N/F262Y/C264S +++ 795/796F262Y/C264S ++ 797/798 C264S/I291V + 799/800 Y24E/C264S/A294V + 801/802S28N + 803/804 S28N/K423E + 805/806 Y24E/S28N/F262Y + 807/808Y24E/S28N + ^(a)Levels of increased production were determined relativeto the reference polypeptide of SEQ ID NO: 770, and defined as follows:“+” = production at least 1.2-fold, but less than 3-fold; “++” = atleast 3-fold, but less than 6-fold; “+++” = at least 6-fold, but lessthan 9-fold; and “++++” = at least 9-fold increased production, ascompared to the reference polypeptide.

TABLE 15.2 Saturation Mutagenesis Round 2 Variants and RebD Levels SEQID NO: Amino Acid Differences (nt/aa) (Relative to SEQ ID NO: 770)Increased RebD^(a) 809/810 Y24V ++++ 811/812 C264A ++++ 813/814 Y24L +++815/816 S28L ++ 817/818 Y269S ++ 819/820 H366Q ++ 821/822 H366T ++823/824 S28G + 825/826 N32C + 827/828 K325G + 829/830 Y269W + 831/832N32S + 833/834 M351L + 835/836 H366L + 837/838 N32R + 839/840 C264G +841/842 K325H + 843/844 F341V + 845/846 S28K + ^(a)Levels of increasedproduction were determined relative to the reference polypeptide of SEQID NO: 770 and defined as follows: “+” = production that of thereference polypeptide, but less than 2-fold; “++” = at least 2-fold, butless than 3-fold; “+++” = at least 3-fold, but less than 4-fold; and“++++” = at least 4-fold increased production, as compared to thereference polypeptide.

HTP Assay for Glucosylation of Rebaudioside A

The remaining combinatorial round 2 libraries were screened as follows:Assays were performed on 96-well plates of cleared E. coli culturelysates expressing SEQ ID NO: 769 variants with lysate loading of 25 μLlysate in 100 μL reactions or with 10 μL4-fold diluted lysate in 100 μLreactions with substrate loading of 1 mM rebaudioside A (Sigma, >96%purity) and co-substrate loading of 1 mM UDP-glucose (Sigma, >98%purity). The following reaction conditions were used: 50 mM Tris-HClbuffer, pH 7.5, 3 mM MgCl₂, 40° C. in a Thermotron® titre-plate shakerwith 300 RPM shaking for 1-2 h. The reactions were either quenched with0.5 volume/volume acetonitrile with 0.2% formic acid or 10 μL of thereaction was quenched with 90 μL acetonitrile with 0.2% formic acid andprecipitated by centrifugation. Glycosylated products were detected inthe supernatant by LC-MS/MS following 1:100 or 1:10 dilution in waterwith the instrument and parameters described in Example 1, Table 1.1.Glycosyltransferase variant polypeptides that produced rebaudioside Dfrom rebaudioside A at greater quantities than SEQ ID NO: 770 wereidentified. The engineered polypeptides are listed in Table 15.3.

TABLE 15.3 Additional Combinatorial Round 2 Variants and RebD Levels SEQID NO: Amino Acid Differences Increased (nt/aa) (Relative to SEQ ID NO:770) RebD^(a) 847/848 I56T/F70L/N191D/M246K/L354I/M365I +++ 849/850H69Q/F70L/N191D/M246K/L354I/M365I +++ 851/852 P175S/M246K/L3541 +++853/854 P175S +++ 855/856 I56T/F70L/P175S/M246K/L354I ++ 857/858I56T/H69Q/P175S/N191D/M246K/L354I ++ 859/860I56T/F70L/P175S/N191D/L354I/M365I ++ 861/862 I56T/F70L/N191D/L354I ++863/864 P175S/N191D ++ 865/866 P175S/L354I ++ 867/868I56T/H69Q/F70L/M246K/L354I ++ 869/870 F70L/N191D/M246K/L354I/M365I ++871/872 I56T/H69Q/F70L/P175S/L354I ++ 873/874 P175S/M246K/L354I/M365I ++875/876 P175S/N191D/M246K/L354I/M365I ++ 877/878H69Q/P175S/N191D/M246K/L354I ++ 879/880 P175S/N191D/L354I ++ 881/882F70L/P175S/N191D/M246K/L354I/M365I ++ 883/884 H69Q/F70L/L354I/M365I ++885/886 I56T/H69Q/M246K/L354I ++ 887/888 H69Q/P175S/L354I + 889/890I56T/H69Q/F70L/N191D/M246K/L354I/M365I + 891/892I56T/H69Q/F70L/P175S/N191D/M246K/L354I + 893/894 I56T/P175S/M246K +895/896 I56T/P175S/L354I/M365I + 897/898 I56T/H69Q/P175S/M246K/L354I +899/900 I56T/N191D/M246K/L354I + 901/902 L354I/M365I + 903/904M246K/L354I + 905/906 I56T/P175S/L354I + 907/908I56T/F70L/P175S/M246K/M365I + 909/910 I56T/M246K + 911/912I56T/H69Q/M246K/M365I + 913/914 I126F/C220L + 915/916I56T/H69Q/P175S/M246K/L354I/M365I + 917/918 I126F/K403R + 919/920I56T/M246K/L354I/M365I + 921/922 I56T/L354I + 923/924F70L/N191D/M246K/M365I + 925/926 H69Q/L354I + 927/928I56T/H69Q/F70L/P175S/N191D/M246K + 929/930 H69Q/M246K/L354I/M365I +931/932 I126F + 933/934 H69Q + 935/936H69Q/F70L/P175S/N191D/M246K/L354I/ + M365I 937/938P175S/N191D/L354I/M365I + 939/940 F70L + 941/942 N191D/M246K/L354I +943/944 F70L/P175S/N191D/L354I/M365I + 945/946I56T/F70L/P175S/N191D/M246K/L354I + 947/948 H69Q/F70L/M246K + 949/950L354I + 951/952 I56T/F70L/P175S/N191D/M246K + ^(a)Levels of increasedproduction were determined relative to the reference polypeptide of SEQID NO: 770, and defined as follows: “+” = production at least 1.5-fold,but less than 3-fold; “++” = at least 3-fold, but less than 4.5-fold;and “+++” = at least 4.5-fold increased production, as compared to thereference polypeptide.

HTP Assay for Glucosylation of Rebaudioside I

88 variants from the round 2 saturation mutagenesis library werescreened as follows: Assays were performed on 96-well plates of clearedE. coli culture lysates expressing SEQ ID NO: 769 variants with lysateloading of 25 μL lysate in 100 μL reactions with substrate loading of 1mM rebaudioside I (prepared from rebaudioside A as described in example10) and co-substrate loading of 1 mM UDP-glucose (Sigma, >98% purity).The following reaction conditions were used: 50 mM potassium phosphatebuffer, pH7, 3 mM MgCl₂, 40° C. in a Thermotron® titre-plate shaker with300 RPM shaking for 66 h. The reactions were quenched by adding 10 μL ofthe reaction to 90 μL acetonitrile with 0.2% formic acid andprecipitated by centrifugation. Glycosylated products were detected inthe supernatant by LC-MS/MS following 1:10 dilution in water with theinstrument and parameters described in Table 15.5. Glycosyltransferasevariant polypeptides that produced rebaudioside M from rebaudioside I atgreater quantities than SEQ ID NO: 770 were identified. The top twoengineered polypeptides are SEQ ID NO: 1292 and 1294, which havemutations F156R and G199H, respectively, relative to SEQ ID NO: 770.

Shake-flask scale cultures were grown for SFP production as described inExample 1 for analysis of variants with the amino acid mutations shownin Table 15.4 (relative to SEQ ID NO: 770).

SFP Characterization Assay and Analysis for Glucosyl Transfer fromADP-Glucose to Rebaudioside A

Shake flask powders were reconstituted to provide 20 g/L powder. Then,10 μL of these stocks were diluted in 100 μL total reaction volume of 50mM Tris-HCl buffer, pH 7.5, with 3 mM MgCl₂, 1 mM rebaudioside A(Sigma, >96% purity), and 4 mM ADP-glucose (Sigma, >93% purity). Thereaction was performed at 30° C. in a Thermotron® titre-plate shakerwith 300 RPM shaking for 0-19 h. The reaction was quenched andprecipitated as described in Example 14. Glycosylated products weredetected in the supernatant by LC-MS/MS following 1:100 dilution inwater with the instrument and parameters described in Example 1, Table1.1.

TABLE 15.4 Shake Flask Powder Characterization of Round 2 Variants andRebD Levels SEQ ID NO: Amino Acid Differences (nt/aa) (Relative to SEQID NO: 770) Increased RebD^(a) 789/790 Y24E/S28N/F262Y/C264S +++ 791/792Y24E/S28N/F262Y/C264S/K423E +++ 793/794 S28N/F262Y/C264S ++ 795/796F262Y/C264S ++ ^(a)Levels of increased production were determinedrelative to the reference polypeptide of SEQ ID NO:770, at 0.5 h anddefined as follows: “−” = production less than the referencepolypeptide; “+” = production at least that of the referencepolypeptide, but less than 5-fold; “++” = at least 5-fold, but less than10-fold; and “+++” = at least 10-fold increased production, as comparedto the reference polypeptide.

All variants in Table 15.4 (i.e., variants of SEQ ID NOS: 790, 792, 794,and 796) produced rebaudioside D from rebaudioside A with ADP-glucose,at greater quantities than SEQ ID NO: 770. Thus, these engineeredADP-glycosyltransferase enzymes provide new biocatalytic reagents forthe β-glucosylation of rebaudioside A to rebaudioside D. In theseexperiments, the variant of SEQ ID NO: 792, had the highest initialactivity on rebaudioside A with ADP-glucose as a co-substrate. Thus, theencoding polynucleotide (SEQ ID NO: 791) was selected for furtherdirected evolution.

TABLE 15.5 HPLC-MS/MS Analysis of Steviol Glycosides Instrument AgilentHPLC 1200 series, Sciex 4000 QTrap Column Poroshell 120 EC C18 50 × 3.0mm, 2.7 μm with Poroshell 120 EC C18 5 × 3.0, 2.7 μm guard column(Agilent Technologies) Mobile phase Gradient (A: 0.1% formic acid inwater, B: 0.1% formic acid in methanol) Time (m) % B 0 40 0.50 53 5.0053 5.50 70 7.50 70 8.00 95 8.50 95 8.51 60 9.20 40 Flow rate 0.8 mL/mRun time 9.2 m Peak retention Rebaudioside M: 4.37 m times RebaudiosideI: 6.70 m Other glucosylated rebaudioside I product: 4.8 m Secondglucosylated rebaudioside I product: 6.7 m Column 40° C. temperatureInjection volume  10 μL MS detection MRM 990/828 (for stevioltetraglycosides, e.g., rebaudioside A), 1152/828 (for steviolpentaglycosides, e.g., rebaudioside D), 1314/828 (steviolhexaglycosides, e.g., rebaudioside M), 828/666 (for stevioltriglycosides, e.g., stevioside), 666/504 (steviol diglycosides, e.g.,rubusoside) MS conditions MODE: MRM; CUR: 30; IS: 4750; CAD: high; TEM:550° C.; GS1: 50; GS2: 50; DP: 150; EP: 10; CXP: 14; DT: 50 ms for eachtransition. For the first three transitions CE: 85; for the last twotransitions CE: 60.

Example 16 ADP-Glycosyltransferase Variants of SEQ ID NO: 792

In this Example, experiments for evolution and screening of GTpolypeptides derived from SEQ ID NO: 792 for improved glucosylation ofsteviol glycosides using ADP-glucose are described. Directed evolutionof the GT encoded by SEQ ID NO: 791 (i.e., SEQ ID NO: 792) was carriedout by constructing libraries of variant genes in which mutationsassociated with improved activity in previous rounds above wererecombined. These libraries were then plated, grown, and screened usingthe high-throughput (HTP) assay described below to provide a third round(“Round 3”) of engineered GT variant polypeptides withglucosyltransferase activity toward ADP-glucose and steviol glycosides.

HTP Assay for Glucose Transfer from ADP-Glucose to Rebaudioside A

Assays were performed on 96-well plates of cleared E. coli culturelysates expressing SEQ ID NO: 792 variants with lysate loading of 25 μLlysate in 100 μL reactions and with substrate loading of 1 mMrebaudioside A (>97% purity) and co-substrate loading of 1 mMADP-glucose (Sigma, >93% purity). The following reaction conditions wereused: 50 mM KPhos buffer, pH 7, 3 mM MgCl₂, 40° C. in a Thermotron®titre-plate shaker with 300 RPM shaking for 5-6 h. Then, 10 μL of thereactions were quenched with 90 μL acetonitrile with 0.2% formic acid,centrifuged 10 m at 4° C., and, the supernatants were analyzed byRapidFire-MS/MS as described in Example 9, Table 9.3.Glycosyltransferase variant polypeptides that produced rebaudioside Dfrom rebaudioside A with ADP-glucose, at greater quantities than SEQ IDNO: 792 were identified. The engineered polypeptides are listed in Table16.1. Shake-flask scale cultures were grown for SFP production asdescribed in Example 1 for analysis of variants shown in Table 16.1relative to SEQ ID NO: 792.

TABLE 16.1 Round 3 Variants and RebD Levels SEQ ID NO: Amino AcidDifferences Increased (nt/aa) (Relative to SEQ ID NO: 792) RebD^(a)953/954 F70L/I126F/N191D/M246K/K325H/M351L/L354I/H366Q/E423K ++++955/956 F70L/I126F/N191D/M246K/K325H/M351L/H366Q/E423K ++++ 957/958F70L/I126F/N191D/M246K/L354I/H366Q +++ 959/960F70L/I126F/P175S/N191D/M246K/K325H/L354I/H366Q +++ 961/962F70L/I126F/M246K/L354I/H366Q +++ 963/964F70L/I126F/N191D/M246K/K325H/M351L/L354I/E423K +++ 965/966F70L/I126F/M246K/P330Q/H366Q +++ 967/968F70L/I126F/P175S/K325H/P330Q/M351L/L354I/H366Q +++ 969/970E24L/N32S/Y269S/A382G/I385V/D389E/L402I/I406M ++ 971/972 E24L/N32S/P330Q++ 973/974 E24L/N32S/S264A/Y269S/P330Q/L402V/K403R ++ 975/976E24L/N32S/S264A/P330Q ++ 977/978 I126F/T211E/T260V/E423K ++ 979/980I126F/C220L/T260V ++ 981/982 E24L/N32S/Y269S/K403R ++ 983/984E24L/N32S/Y269S ++ 985/986 I126F/C220L/I316V ++ 987/988E24L/N32S/S264A/Y269S/A382G/K403R/I406M ++ 989/990 C220L/T260V/E423K ++991/992 I126F/T211E/C220L/G253D/I316V/I342L ++ 993/994I126F/T211E/C220L/D275Q/V279L/L323V ++ 995/996 C220L/T260V + 997/998I56V/C220L/T260V/E423K + 999/1000 E24L/S264A/Y269S/D389E/L402V/I406M +^(a)Levels of increased production were determined relative to thereference polypeptide of SEQ ID NO: 792, and defined as follows: “+” =production at least 1.4-fold that of the reference polypeptide, but lessthan 2.8-fold; “++” = at least 2.8-fold, but less than 4.2-fold; “+++” =at least 4.2-fold, but less than 5.6-fold; and “++++” = at least5.6-fold increased production, as compared to the reference polypeptide.SFP Characterization Assay and Analysis for Glucosyl Transfer fromADP-Glucose to Rebaudioside A

Shake flask powders were reconstituted to provide 20 g/L powder. Then,10 μL of these stocks were diluted in 100 μL total reaction volume of 50mM KPhos buffer, pH7, with 3 mM MgCl₂, 1 mM rebaudioside A (>97%purity), and 2 mM ADP-glucose (Sigma, >93% purity).

The reaction was performed at 40° C. in a Thermotron® titre-plate shakerwith 300 RPM shaking for 0-21 h. The reaction was quenched andprecipitated as described above. Glycosylated products were detected inthe supernatant by LC-MS/MS following 1:10 dilution in water with theinstrument and parameters described in Example 1, Table 1.1.

TABLE 16.2 Shake Flask Powder Characterization of Round 3 Variants andRebD Levels SEQ ID NO: Amino Acid Differences Increased (nt/aa)(Relative to SEQ ID NO: 792) RebD^(a) 953/954F70L/I126F/N191D/M246K/K325H/M351L/L354I/H366Q/E423K +++ 955/956F70L/I126F/N191D/M246K/K325H/M351L/H366Q/E423K ++ 989/990C220L/T260V/E423K +++ ^(a)Levels of increased production were determinedrelative to the reference polypeptide of SEQ ID NO: 792, at 2 h anddefined as follows: “+” = production at least that of the referencepolypeptide, but less than 2-fold; “+” = production at least 2-fold, butless than 3-fold; and “+++” = at least 3-fold increased production, ascompared to the reference polypeptide.

In these experiments, all of the variants in Table 16.2 (i.e., SEQ IDNOS: 954, 956, and 990) produced rebaudioside D from rebaudioside A withADP-glucose, at greater quantities than SEQ ID NO: 792. The variant ofSEQ ID NO:954 had the highest activity on rebaudioside A withADP-glucose as a co-substrate in these experiments. Thus, the encodingpolynucleotide (SEQ ID NO: 953) was selected for further directedevolution.

Example 17 ADP-Glycosyltransferase Variants of SEQ ID NO: 954 forSteviol Glycoside Glucosylation, Including Glucosylation of RebaudiosideI to Rebaudioside M

In this Example, experiments for evolution and screening of GTpolypeptides derived from SEQ ID NO: 954 for improved glucosylation ofsteviol glycosides using ADP-glucose are described. Directed evolutionof the GT encoded by SEQ ID NO: 953 (i.e., SEQ ID NO: 954) was carriedout by constructing libraries of variant genes in which mutationsassociated with improved activity in previous rounds above wererecombined. These libraries were then plated, grown, and screened usingthe high-throughput (HTP) assay described below to provide a fourthround (“Round 4”) of engineered GT variant polypeptides withglucosyltransferase activity toward ADP-glucose and steviol glycosides.

HTP Assay for Glucose Transfer from ADP-Glucose to Rebaudioside A

Cells were lysed with 400 μL lysis buffer as described in Example 13.Assays were performed on 96-well plates of cleared E. coli culturelysates expressing SEQ ID NO: 954 variants with lysate loading of 20 μLlysate in 100 μL reactions and with substrate loading of 1 mMrebaudioside A (>97% purity) and co-substrate loading of 1 mMADP-glucose (Sigma, >93% purity). The following reaction conditions wereused: 50 mM KPhos buffer, pH 7, 3 mM MgCl₂, 40° C. in a Thermotron®titre-plate shaker with 300 RPM shaking for 4h. The reactions werequenched as described in Example 16, and the supernatants were analyzedby RapidFire-MS/MS as described in Example 9, Table 9.3.Glycosyltransferase variant polypeptides that produced rebaudioside Dfrom rebaudioside A with ADP-glucose, at greater quantities than SEQ IDNO: 954 were identified. The engineered polypeptides are listed in Table17.1. Shake-flask scale cultures were grown for SFP production asdescribed in Example 1 for analysis of variants with the following aminoacid mutations shown in Table 17.1 (relative to SEQ ID NO: 150).

TABLE 17.1 Round 4 Variants and RebD Levels SEQ ID NO: Amino AcidDifferences Increased (nt/aa) (Relative to SEQ ID NO: 954) RebD^(a)1001/1002 P175S/T211E/S264A/V279L/I316V/L323V ++++ 1003/1004T211E/1385V/D389E ++++ 1005/1006 I56V/T211E/S264A/I316V/D389E +++1007/1008 I56V/P175S/A197P/G253D/I385V/D389E +++ 1009/1010A197P/T211E/L402I +++ 1011/1012 T211E/L4021/K403R +++ 1013/1014P175S/T211E/V279L/L323V/P330Q/L402I/K403R/I406M ++ 1015/1016P175S/A197P/T211E/S264A/P330Q ++ 1017/1018P175S/T211E/L323V/A382G/L402I/K403R/1406M ++ 1019/1020P175S/T211E/K403R/1406M ++ 1021/1022I56V/P175S/A197P/T211E/P330Q/A382G/1385V + 1023/1024P175S/S264A/L323V/P330Q + 1025/1026 A197P/V279L/L323V + 1027/1028N162R/C220L/W226V/N367W + 1029/1030 I56V/S264A/I385V/D389E + 1031/1032E24L/N32S/F126A/E198P/M201G/N367W + 1033/1034I56V/T211E/V279L/L323V/P330Q/L402I + 1035/1036N32S/H97G/E198P/Q202G/W226V/T260V + 1037/1038E24L/N32S/E198P/M201G/W226V + 1039/1040I56V/P175S/S264A/A382G/I385V/D389E/L402V/I406M + 1041/1042E198P/M201G/N367W + 1043/1044 E24L/N32S/L146A/VV226V + 1045/1046 D389E +1047/1048 M201G/Q202G/N367W + 1049/1050 T211E/A382G/I406M + 1051/1052E24L/N32S/E198P/M201G/C220L/W226V + 1053/1054 H97G/Q202G/N367W +1055/1056 E24L/H97G/T260V/N367W + 1057/1058P175S/S264A/I316V/L323V/P330Q/K403R/1406M + 1059/1060A197P/T211E/I316V/I342L/1406M + 1061/1062 N32S/Q202G/N367W + 1063/1064A197P/T211E/I316V/A382G/D389E/L402I/K403R + 1065/1066 N32S/W226V/N367W +1067/1068 I56V/T211E/G253D/1316V/L323V + 1069/1070E24L/N32S/F126A/E198P/Q202G/C220L/W226V/T260V/Y269S + 1071/1072E198P/Q202G/C220L/Y269S/N367W + 1073/1074P175S/S264A/1316V/D389E/L402V/ + 1075/1076 E24L/N32S/H97G/N162R/Q202G +1077/1078 I56V/A197P/S264A/V279L/P330Q/A382G/D389E/L402V/K403R/I406M +^(a)Levels of increased production were determined relative to thereference polypeptide of SEQ ID NO: 954, and defined as follows: “+” =production at least 1.3-fold, but less than 2.6-fold; “++” = at least2.6-fold, but less than 3.9-fold; “+++” = at least 3.9-fold, but lessthan 5.2-fold; and “++++” = at least 5.2-fold increased production, ascompared to the reference polypeptide.SFP Characterization Assay and Analysis for Glucosyl Transfer fromADP-Glucose to Rebaudioside A

Shake flask powders were reconstituted to provide 50 g/L powder. Then, 1μL of these stocks were diluted in 100 μL total reaction volume of 50 mMKPhos buffer, pH7, with 3 mM MgCl₂, 1 mM rebaudioside A (>97% purity),and 2 mM ADP-glucose (Sigma, >93% purity). The reaction was performed at40° C. in a Thermotron® titre-plate shaker with 300 RPM shaking for 2h.The reaction was then diluted 1:5 in water, and 25 μL of the dilutedreaction was quenched with 75L acetonitrile with 0.2% formic acid andprecipitated by centrifugation. Glycosylated products were detected inthe supernatant by RapidFire-MS/MS following 1:10 dilution in water withthe instrument and parameters described in Example 9, Table 9.3.

TABLE 17.2 Shake Flask Characterization of Round 4 Variants and RebDLevels SEQ ID NO: Amino Acid Differences Increased (nt/aa) (Relative toSEQ ID NO: 954) RebD^(a) 1053/1054 H97G;Q202G/N367W +++ 1031/1032E24L/N32S/F126A/E198P/M201G/N367W +++ 1027/1028 N162R/C220L/W226V/N367W++ 1051/1052 E24L/N32S/E198P/M201G/C220L/W226V ++ 1057/1058P175S/S264A/I316V/L323V/P330Q/K403R/ ++ I406M 1013/1014P175S/T211E/V279L/L323V/P330Q/L402I/ + K403R/I406M 1001/1002P175S/T211E/S264A/V279L/I316V/L323V + 1029/1030 I56V/S264A/I385V/D389E −^(a)Levels of increased production were determined relative to thereference polypeptide of SEQ ID NO: 954, and defined as follows: “−” =production less than that of the reference polypeptide; “+” = productionat least that of the reference polypeptide, but less than 1.5-fold; “++”= at least 1.5-fold, but less than 2-fold; and “+++” = at least 2-foldincreased production, as compared to the reference polypeptide.

With the exception of SEQ ID NO: 1030, all other variants in Table 17.2(SEQ ID NOS: 1054, 1032, 1028, 1052, 1058, 1014, and 1002) producedrebaudioside D from rebaudioside A with ADP-glucose, at greaterquantities than SEQ ID NO: 954, in these experiments. The variant withmutations H97G, Q202G, and N367W (SEQ ID NO: 1054), relative to SEQ IDNO: 954, had the highest activity on rebaudioside A with ADP-glucose asa co-substrate in these experiments. Thus, the encoding polynucleotide(SEQ ID NO: 1053) was selected for further directed evolution.

SFP Characterization Assay and Analysis for Glucosyl Transfer fromUDP-Glucose to Rebaudioside I

Shake flask powders were reconstituted to provide 50 g/L powder. Then, 2L of these stocks were diluted in 100 μL total reaction volume of 50 mMKPhos buffer, pH 7, with 3 mM MgCl₂, 1 mM rebaudioside A (>97% purity),and 2 mM UDP-glucose (Sigma, >98% purity). The reaction was performed at40° C. in a Thermotron® titre-plate shaker with 300 RPM shaking for 20h. The reaction was then quenched with 0.5 volume/volume acetonitrilewith 0.2% formic acid and precipitated by centrifugation. Glycosylatedproducts were detected in the supernatant by LC-MS/MS following 1:20dilution in water with the instrument and parameters described inExample 15, Table 15.5.

TABLE 17.3 Shake Flask Characterization of Round 4 Variants and RebMLevels SEQ ID NO: Amino Acid Differences Increased (nt/aa) (Relative toSEQ ID NO: 954) RebM^(a) 1001/1002 P175S/T211E/S264A/V279L/I316V/L323V++ 1013/1032 E24L/N32S/F126A/E198P/M201G/N367W + 1057/1058P175S/S264A/I316V/L323V/P330Q/K403R/ + I406M 1013/1014P175S/T211E/V279L/L323V/P330Q/L402I/ − K403R/I406M 1020/1030I56V/S264A/I385V/D389E − 1051/1052 E24L/N32S/E198P/M201G/C220L/W226V −1053/1054 H97G/Q202G/N367W − 1027/1028 N162R/C220L/W226V/N367W −^(a)Levels of increased production were determined relative to thereference polypeptide of SEQ ID NO: 954, and defined as follows: “−” =production less than that of the reference polypeptide; “+” = productionat least that of the reference polypeptide, but less than 1.2-fold; and“++” = at least 1.2-fold increased production, as compared to thereference polypeptide.

SEQ ID NO: 954 and all variants in Table 17.3 (SEQ ID NOS: 1054, 1032,1028, 1052, 1014, 1002, and 1030) produced rebaudioside M fromrebaudioside I with UDP-glucose, at levels above the negative control.SEQ ID NOS: 1002, 1032, and 1058 produced rebaudioside M fromrebaudioside I at levels equal to or above SEQ ID: 954 levels withUDP-glucose. The variant with mutations P175S, T211E, S264A, V279L,I316V, and L323V (SEQ ID NO: 1002), relative to SEQ ID NO: 954, had thehighest activity on rebaudioside I with UDP-glucose as a co-substrate.Thus, the encoding polynucleotide (SEQ ID NO: 1001) was selected forfurther directed evolution for the transformation of rebaudioside I torebaudioside M.

Example 18 Sucrose Synthase Variants of SEQ ID NO: 74

In this Example, experiments for the evolution and screening of sucrosesynthase (SuS) polypeptides derived from SEQ ID NO: 73 for improvedproduction of ADP-glucose from sucrose and ADP are described. Directedevolution of the SuS encoded by SEQ ID NO: 73 (i.e., SEQ ID NO: 74) wascarried out by constructing libraries of variant genes in whichpositions associated with certain structural features of the enzyme weresubjected to saturation mutagenesis and diversity from homologs inpublically available databases was recombined. These libraries were thenplated, grown, and screened using the high-throughput (HTP) assaydescribed below to provide a first round (“Round 1”) of 37 engineeredSuS variant polypeptides with improved activity toward synthesizingADP-glucose.

HTP Assay for Glucose Transfer from Sucrose to ADP

Assays were performed on 96-well plates of cleared E. coli culturelysates expressing SEQ ID NO: 73 variants with lysate loading of 25 μLlysate in 100 μL reactions and with substrate loading of 30% w/v sucrose(Sigma) from a 60% stock solution in water and co-substrate loading of 2mM ADP (Sigma, >95%). The following reaction conditions were used: 50 mMTris-HCl buffer, pH 7.5, 3 mM MgCl₂, 30° C. in a Thermocycler for 2 h.The reactions were heat quenched at 95° C. for 10 minutes, and thenanalyzed by a colorimetric D-fructose dehydrogenase assay adapted fromthe literature (See e.g., Ameyama et al., J. Bacteriol., 145:814-823[1981]; and Ameyama, Meth. Enzymol., 89:20-29 [1982]). Briefly, anovernight enzyme-coupled assay was conducted in 96-well plates with 20μL sample, diluted such that fructose concentration is <1 g/L, 20 μL 100mM potassium ferricyanide (Sigma P-8131), and 160 μL 0.8 units/mLfructose dehydrogenase (Sigma F4892) dissolved in pH 4.6 McIlvainebuffer with 0.1% Triton X-100. This reaction quantitatively convertsfructose to K₄Fe(CN)₆, which is then quantified colorimetrically byadding 67 μL of the overnight reaction to 33 μL of stop solution (0.3%w/v sodium dodecyl sulfate, Sigma L-4509, 8.1% v/v phosphoric acid,Sigma P-6560, and 0.5% w/v ferric sulfate, Sigma F-1135) and shaking for20 minutes to allow for complete conversion of K₄Fe(CN)₆ to Prussianblue, the absorbance of which is read on a plate reader at a wavelengthof 690 nm.

Following the primary assay, 84 engineered sucrose synthase (SuS)variant polypeptides with higher fructose, and therefore higherstoichiometric ADP-glucose, formation activity than SEQ ID NO: 74 werescreened in triplicate at a lower substrate load of 2% w/v sucrose(Sigma) and co-substrate load of 1 mM ADP (Sigma, >95%). The engineeredpolypeptides are listed in Table 18.1.

TABLE 18.1 Round 1 SuS Variants and Fructose Levels SEQ ID NO: AminoAcid Differences Increased (nt/aa) (Relative to SEQ ID NO:74)Fructose^(a) 1079/1080 A4E/Y33Q/L47H/A52D/V343H/F532S +++ 1081/1082A4E/L47H/A52D/F532S +++ 1083/1084 A4E/I113Q/F532S ++ 1085/1086V343S/F532S ++ 1087/1088 R615L/A789D ++ 1089/1090 Q7T ++ 1091/1092 R722Y++ 1093/1094 R615E ++ 1095/1096 A4E/L9T/Q349H/F532S ++ 1097/1098A4E/V343H ++ 1099/1100 A4E/P13R/I113Q ++ 1101/1102 R221H ++ 1103/1104A4E/P13R/I113Q/V343H/F532S + 1105/1106 A4E/P13R/F532S + 1107/1108 R44K +1109/1110 A4E/P13R/I113Q/F532S + 1111/1112 A4E/Y33Q/I113Q + 1113/1114V695L + 1115/1116 Q8M + 1117/1118 G117D/R440T + 1119/1120 R440T +1121/1122 H788E + 1123/1124 R478T + 1125/1126 R611V + 1127/1128 R615T +1129/1130 Q95S + 1131/1132 F532R + 1133/1134 Q444K + 1135/1136 R440P +1137/1138 R478V + 1139/1140 R615C + 1141/1142 F532T + 1143/1144 Q444T +1145/1146 R136S + 1147/1148 R583Q + 1149/1150 R615V + 1151/1152 R221A +^(a)Levels of increased production were determined relative to thereference polypeptide of SEQ ID NO: 74, and defined as follows: “+” =production at least that of the reference polypeptide, but less than1.5-fold; “++” = at least 1.5-fold, but less than 2.5-fold; and “+++” =at least 2.5-fold increased production, as compared to the referencepolypeptide.

Example 19 Sucrose Synthase Variants of SEQ ID NO: 1080

Directed evolution of the SuS encoded by SEQ ID NO:1079 (i.e., SEQ IDNO:1080) was continued by constructing libraries of variant genes inwhich mutations associated with improved activity above were recombinedand libraries in which additional homolog diversity was recombined.These libraries were then plated, grown, and screened using thehigh-throughput (HTP) assay described in Example 18, with the lowersubstrate load, to provide a second round (“Round 2”) of 34 engineeredSuS variant polypeptides with activity towards the generation ofADP-glucose.

Following primary screening, 42 variants were retested in duplicate and56 variants were retested in triplicate under the same conditions withthe following modifications: 50 mM Tris-HCl pH 7.5 was changed to 50 mMpotassium phosphate buffer, pH 7 and the temperature was increased to50′C. The fructose dehydrogenase assay described in Example 18, was usedto quantify fructose production as a proxy for stoichiometricADP-glucose. The resulting engineered polypeptides are listed in Table19.1. Shake-flask scale cultures were grown for protein characterizationas described in Example 1 for variants with the amino acid mutationsshown in Table 19.1 (relative to SEQ ID NO: 1080).

TABLE 19.1 Round 2 SuS Variants and Fructose Levels SEQ ID NO: AminoAcid Differences Increased (nt/aa) (Relative to SEQ ID NO: 1080)Fructose^(a) 1153/1154 R136S/R440P/Q444T/R478T/G603S ++ 1155/1156V68A/R129E/S248A/I595V/V6001I/I756V ++ 1157/1158 I113L/A225E/R266N/R415K++ 1159/1160 V68A/R129E/S248A ++ 1161/1162M75V/A105E/R154H/I215F/I264V/A345T ++ 1163/1164 A225E/I372V/E534H ++1165/1166 R85V/I170L/A225E/R266N/E534H ++ 1167/1168Q95S/Q444T/R478V/G603S/M792S ++ 1169/1170 H47L/R221H ++ 1171/1172I87E/T125E/N230D/I267V/W375Y/I464F/ ++ T708A 1173/1174R440P/Q444T/R583Q/K724H/H788E + 1175/1176 Q95S/R478T/K724H + 1177/1178R266N + 1179/1180 R136S/R440T/Q444T/R478V/R583Q/H788E + 1181/1182R440T/R478V + 1183/1184 R306L/P358E/N703Y/Q776E + 1185/1186R136S/Q444T/R478V/R583Q/H788E/M792S + 1187/1188 L98V/S250D + 1189/1190Q95S/R440T/R478V/K724H/H788E/M792S + 1191/1192Q95S/Q201E/R478V/R583Q/K724H/H788E + 1193/1194 R93V/D477K/A635S +1195/1196 M75V/A105E/A345T/T410S/Q769R + 1197/1198 M75V/A105E/P530L +1199/1200 M75V/A345T/P530L + 1201/1202 Q8M/R221H + 1203/1204 V466I +1205/1206 Q95S/R136S/H788E + 1207/1208 P358E/L636Q/V737I + 1209/1210R93V/R129E/S506P/R550H/I595V/A719C/ + I756V 1211/1212V68A/G189R/I272L/V316I/D477K/A719C/ + I756V 1213/1214V126L/V314L/N499H/D549E/G589E/R755G + 1215/1216Q95S/Q444T/R478V/K724H/H788E + 1217/1218 Q95S/R385L/R478V/R583Q/H788E +1219/1220 V68A/D146N/S248A/V387I/S506P/R550H + ^(a)Levels of increasedproduction were determined relative to the reference polypeptide of SEQID NO: 1080, and defined as follows: “+” = production at least that ofthe reference polypepide, but less than 1.2-fold; and “++” = at least1.2-fold increased production, as compared to the reference polypeptide.Characterization of SuS Shake Flask Powders with a Coupled Assay forGlucosylation of Rebaudioside A Using ADP-glucose Produced by SuS fromSucrose and ADP

First, SuS lyophilized shake flask powders were reconstituted in waterand added for final concentrations ranging from 0.025-1 g/L of proteinin total reaction volume. The reaction conditions were as follows: 50 mMpotassium phosphate buffer, pH 7, 3 mM MgCl₂, with 1 mM rebaudioside A(>97% purity), 10 mM sucrose (Sigma), and 1 mM ADP (Sigma). The GT addedto catalyze the glucosyl transfer from ADP-glucose to rebaudioside A wasSEQ ID NO: 548 at 2 g/L final concentration in 100 μL total reactionvolume. The reaction was performed at 50° C. in a Thermotron®titre-plate shaker with 300 RPM shaking for 1 h. The reaction describedabove was quenched by adding 10 μL assay to 90 μL acetonitrile with 0.2%formic acid and precipitated by centrifugation. Glycosylated productswere detected in the supernatant by RapidFire SPE-MS/MS following 1:10dilution in water with the instrument and parameters described inExample 9, Table 9.3. The relative levels of rebaudioside I producedfrom rebaudioside A in the coupled shake flask powder assays are listedin Table 19.2.

TABLE 19.2 Round 2 Shake Flask Powder Characterization in Coupled AssaySEQ ID NO: Amino Acid Differences (nt/aa) (Relative to SEQ ID NO: 1080)RebI Levels^(a) 1161/1162 M75V/A105E/R154H/I215F/I264V/A345T ++1153/1154 R136S/R440P/Q444T/R478T/G603S + 1165/1166R85V/I170L/A225E/R266N/E534H + 1157/1158 I113L/A225E/R266N/R415K +1163/1164 A225E/I372V/E534H − ^(a)Levels of increased production weredetermined relative to the reference polypeptide of SEQ ID NO: 1080, anddefined as follows: “−” = production less than 0.8-fold; “+” =production at least 0.8-fold, but less than 1.2-fold; and “++” = atleast 1.2-fold increased production, as compared to the referencepolypeptide.

Example 20 Sucrose Synthase Variants of SEQ ID NO: 1158

Directed evolution of the SuS encoded by SEQ ID NO: 1157 (i.e., SEQ IDNO: 1158) was continued by constructing libraries of variant genes inwhich mutations associated with improved activity above were recombined.These libraries were then plated, grown, and screened using thehigh-throughput (HTP) assay described below, to provide a third round(“Round 3”) of 34 engineered SuS variant polypeptides with activitytowards the generation of ADP-glucose.

HTP Coupled Assay for Glucose Transfer from Sucrose to ADP toRebaudioside A

Libraries were screened using the following high-throughput (HTP) enzymecoupled assay: 10 μL SuS lysate and 2 g/L GT SEQ ID NO: 548 in 100 μLreaction volume with substrate loading of 1 mM rebaudioside A (>97%purity) and co-substrate loadings of 1 mM ADP (Sigma, >95%) and 10 mMsucrose (Sigma). The following reaction conditions were used: 50 mLpotassium phosphate buffer, pH 7, 3 mM MgCl₂, 50° C. in a Thermotron®titre-plate shaker with 300 RPM shaking for 1 h. The reaction describedabove was quenched by adding 10 μL assay to 90 μL acetonitrile with 0.2%formic acid and precipitated by centrifugation. The supernatant wasdiluted 1:10 in water and steviol glycoside products were detected byRapidFire SPE-MS/MS with the instrument and parameters described inExample 9, Table 9.3. After analysis, 56 engineered SuS variantpolypeptides that showed activity coupled with a GT on rebaudioside Awere retested in triplicate with the same conditions and 2.5-foldreduced lysate load. The resulting engineered polypeptides are listed inTable 20.1.

Shake-flask scale cultures were grown for protein characterization asdescribed in Example 1 for variants with the amino acid mutations shownin Table 20.1 (relative to SEQ ID NO: 1158).

TABLE 20.1 Round 3 Variants and RebI Levels SEQ ID NO: Amino AcidDifferences RebI (nt/aa) (Relative to SEQ ID NO: 1158) Levels^(a)1221/1222 R85V/187E/A105E/I215F/I267V/I756V ++++ 1223/1224H47L/R85V/T125E/I372V/R583Q/A635S/1I756V ++++ 1225/1226H47L/R85V/I87E/R154H/I756V ++++ 1227/1228R85V/I87E/R129E/W375Y/I756V/Q776E +++ 1229/1230V68A/R93V/L98V/R136S/S248A/S250D/P358E/R440P/E534H/ +++ K724H 1231/1232H47L/R85V/I87E/A105E/T125E/A635S +++ 1233/1234 H47L/R129E/I170L/A635S+++ 1235/1236 Q8M/R136S/S248A/R478V/H788E +++ 1237/1238H47L/R85V/A105E/R129E/Q201E/N230D/I267V/R583Q ++ 1239/1240H47L/M75V/R85V/A105E/T125E/R129E/I170L/A635S ++ 1241/1242M75V/R85V/R129E/R154H/I264V/W375Y ++ 1243/1244I87E/T125E/R129E/I170L/N230D/I756V ++ 1245/1246H47L/M75V/R85V/I264V/I267V/I372V/K415R/A635S ++ 1247/1248H47L/R85V/I170L/I756V ++ 1249/1250Q8M/Q95S/L98V/R440P/R478V/E534H/V600I/H788E ++ 1251/1252Q8M/V68A/L98V/R221H/S248A/S250D/R440P/D477K/E534H/ ++ I595V/K724H1253/1254 Q8M/V68A/Q95S/L98V/P358E/R478T/I595V/K724H/M792S ++ 1255/1256Q95S/R440P/Q444T/K724H/H788E ++ 1257/1258 I87E/R154H/R306L/W375Y/I756V++ 1259/1260 I170L/I264V/I267V ++ 1261/1262M75V/R85V/I87E/A105E/I264V/I267V/R583Q/T708A ++ 1263/1264Q8M/R93V/Q95S/L98V/R136S/R221H/I595V/V600I/H788E ++ 1265/1266V68A/R93V/Q95S/P358E/R440T/Q444T/R478V/E534H/I595V/G603S ++ 1267/1268H47L/R154H/I372V/W375Y/R583Q/A635S/T708A/I756V ++ 1269/1270H47L/M75V/R85V/A105E/VV375Y/I756V/Q776E ++ 1271/1272R129E/I215F/I372V/I756V ++ 1273/1274 V68A/R93V/Q95S/Q444T/H788E ++1275/1276 H47L/M75V/R85V/I87E/I170L/I372V/I756V ++ 1277/1278Q8M/R93V/Q95S/L113I/S250D/R440T/I595V/V600I/K724H/H788E + 1279/1280H47L/M75V/R85V/I87E/R129E/W375Y/Q776E + 1281/1282R93V/Q95S/L98V/E534H/M792S + 1283/1284 Q8M/V68A/H788E + 1285/1286H47L/T125E/R129E/W375Y/I756V/Q776E + 1287/1288R85V/T125E/I215F/W375Y/K415R/A635S/Q776E + ^(a)Levels of increasedproduction were determined relative to the reference polypeptide of SEQID NO: 1158, and defined as follows: “+” = production at least that ofthe reference polypeptide, but less than 1.5-fold; “++” = production atleast 1.5-fold, but less than 2-fold; “+++” = production at least2-fold, but less than 2.5-fold; and “++++”, at least 2.5-fold increasedproduction, as compared to the reference polypeptide.Characterization of SuS Shake Flask Powders with a Coupled Assay forGlucosylation of Rebaudioside A Using ADP-glucose Produced by SuS fromSucrose and ADP

First, SuS lyophilized shake flask powders were reconstituted in waterand added for final concentrations ranging from 0.025-5 g/L of proteinin total reaction volume. The reaction conditions were as follows: 50 mMpotassium phosphate buffer, pH7, 3 mM MgCl₂, with 1 g/L rebaudioside D,10 mM sucrose (Sigma), and 1 mM ADP (Sigma). The GT added to catalyzethe glucosyl transfer from ADP-glucose to rebaudioside A was SEQ ID NO:561/562 at 1 g/L final concentration in 100 μL total reaction volume.The reaction was performed at 50° C. in a Thermotron® titre-plate shakerwith 300 RPM shaking for 1h. The reaction described above was quenchedby adding 10 μL assay to 90 μL acetonitrile with 0.2% formic acid andprecipitated by centrifugation. Glycosylated products were detected inthe supernatant by RapidFire SPE-MS/MS following 1:10 dilution in waterwith the instrument and parameters described in Example 9, Table 9.3.The relative levels of rebaudioside M produced from rebaudioside Din thecoupled shake flask powder assays are listed in Table 20.2.

TABLE 20.2 Round 3 Shake Flask Powder Characterization in Coupled AssaySEQ ID NO: Amino Acid Differences RebM (nt/aa) (Relative to SEQ ID NO:1158) Levels^(a) 1225/1226 H47L/R85V/I87E/R154H/I756V ++ 1221/1222R85V/187E/A105E/I215F/I267V/I756V ++ 1229/1230V68A/R93V/L98V/R136S/S248A/S250D/P358E/R440P/E534H/K724H + 1227/1228R85V/I87E/R129E/W375Y/I756V/Q776E + 1223/1224H47L/R85V/T125E/I372V/R583Q/A635S/I756V + ^(a)Levels of increasedproduction were determined relative to the reference polypeptide of SEQID NO: 1158, and defined as follows: “+” = production at least that ofthe reference polypeptide, but less than 1.7-fold; and “++” = at least1.7-fold increased production, as compared to the reference polypeptide.

Expression Analysis of SuS Enzymes

The three rounds of engineered SuS polypeptides for ADP recycle withsucrose were analyzed by polyacrylamide gel-electrophoresis to determinerelative protein expression levels. Samples were prepared with 1×LDSloading buffer and 1× reducing agent (Life Technologies). A4-12%Bis-Tris acrylamide gel (Life Technologies) was loaded with 5 μg perlane of lyophilized soluble crude lysate from shake flask scale culturesand run with MES running buffer for 25 min at 200 V, and bands werequantified using ImageJ analysis software. The relative expressionlevels are listed in Table 20.3. SEQ ID NOs: 1079/1080, 1157/1158, and1221/1222 are significantly better expressed genes and/or significantlybetter folded/more stable proteins. These produce more protein than thewild type gene.

TABLE 20.3 Protein Levels of Engineered SuS Variants SEQ ID NO: (nt/aa)Increased Protein^(a) 1079/1080 +++ 1157/1158 ++ 1221/1222 ++ ^(a)Levelsof increased protein were determined relative to the referencepolypeptide of SEQ ID NO: 74, and defined as follows: “+” = bandintensity at least that of the reference polypeptide, but less than2-fold; “++” = band intensity at least 2-fold, but less than 3-fold; and“+++” = at least 3-fold increased band intensity, relative to thereference polypeptide.

Example 21 Transformation of Stevioside to Rebaudioside A with AGT andACSuS

In this Example, experiments conducted to produce rebaudioside A usingAGT and ACSuS are described. The buffer used was 50 m pH7.0 potassiumphosphate with 3 mM MgSO₄. Enzyme and sucrose stock solutions (400 μL)were prepared in the buffer. “Reb A60” was a ˜1:2 mixture of steviosideand rebaudioside A respectively. To a vial under air was added 250 μL of8 μL β-1,3-GT SEQ ID NO: 1290 stock solution, 250 μL of 2 g/L SuS SEQ IDNO: 1158 stock solution and 500 μL of 50 g/L Reb A60 and 2.6 μg/L ADPstock solution in 400 g/L of sucrose. The final compositions was 25 g/LReb A60, 200 g/L sucrose, 2 g/L SEQ ID NO: 1290, 0.5 g/L SEQ ID NO:1158, and 1.3 g/L (3 mM) of ADP. The resulting clear homogenous solutionwas stirred under air (the reaction gradually turned cloudy withformation of precipitates). The progress of the reaction was followed bytaking aliquots and quenched with 1:1 acetonitrile/water. Aftercentrifuging at 4000 rpm/r.t./5 min, the clear homogeneous supernatantwas analyzed by HPLC using the instrument and parameters listed in Table21.1 and Table 21.2. After 48h, the reaction mixture consisted of >90%RebA (Table 21.3).

TABLE 21.1 Analytical HPLC Method Steviol Glycosides Instrument AgilentHPLC 1100 series Column 3.5 × 50 mm 3.5 μm Waters XBridge Phenyl columnMobile phase Isocratic 65:35 A:B A: 0.1% formic acid in water B: 0.1%formic acid in methanol Flow rate  2.4 mL/m Run time   12 m Peakretention times Rebaudioside D: 3.7 m Rebaudioside M: 4.7 m Stevioside:8.8 m Rebaudioside A and I: 9.9 m Column temperature 50° C. Injectionvolume   10 μL UV detection  210 nm

TABLE 21.2 Analytical HPLC Method Steviol Glycosides Instrument ThermoUltimate 3000 UPLC with CAD Column 3.2 × 250 mm 3.5 μm Restek PinnacleII Amino column Mobile phase Isocratic 22:78 A:B A: water B:acetonitrile Flow rate  2.0 mL/m Run time   12 m Peak retention timesFructose: 1.7 m Glucose: 2.0 m Stevioside: 2.6 m Sucrose: 3.1 mRebaudioside A: 4.2 m Rebaudioside I: 6.6 m Rebaudioside D: 10.0 mRebaudioside M: 10.7 m Column temperature 50° C. Injection volume   10μL UV detection  210 nm for organic compounds CAD for sugars

TABLE 21.3 Conversion of Stevioside to Rebaudioside A Over Time t [h] %stevioside % Reb A 0 36 64 1 35 65 2 33 67 4 31 69 8 27 73 24 18 82 48 892 72 6 94

Example 22 Transformation of Stevioside to Rebaudioside A andRebaudioside A to Rebaudioside D with AGT and ACSuS

In this Example, experiments conducted to produce rebaudioside A andrebaudioside D are described. To a vial under air was added 250 μL of 20g/L β-1,3-GT SEQ ID NO: 1290 stock solution, 250 μL of 40 g/L β-1,2-GTSEQ ID NO: 954 stock solution and 500 μL of 20 g/L Reb A 60, 2.6 g/L ADPand 1 g/L SuS SEQ ID NO: 1158 stock solution in 400 g/L of sucrose. Thebuffer used was 50 mM pH 7.0 potassium phosphate with 3 mM MgSO₄. Enzymeand sucrose stock solution (400 g/L) was prepared in the buffer. Thefinal compositions was 10 g/L Reb A 60, 200 g/L sucrose, 5 g/L SEQ IDNO: 1290, 10 g/L SEQ ID NO: 954, 0.5 g/L SEQ ID NO: 1158, and 1.3 g/L (3mM) of ADP. The resulting clear homogenous solution was stirred underair (the reaction gradually turned cloudy with formation ofprecipitates). The progress of the reaction was followed by takingaliquots and quenched with 1:1 acetonitrile/water. After centrifuging at4000 rpm/r.t./5 min, the clear homogeneous supernatant was analyzed byHPLC using the instrument and parameters listed in Table 21.1 and Table21.2. After 24 h, no stevioside remained and formation of 16% of Reb Dwas observed (Table 22.1).

TABLE 22.1 Conversion of Stevioside and Rebaudioside A to Rebaudioside Aand Rebaudioside D t [h] % stevioside % Reb A % Reb D 0 35 65 0 1 27 703 2 25 70 5 4 23 72 5 8 15 79 6 24 0 84 16

Example 23 Transformation of Rebaudioside D to Rebaudioside M with AGTand ACSuS

In this Example, experiments conducted to produce rebaudioside M fromrebaudioside D using AGT and ACSuS variants are described. To a vialunder air was added 100 mg of Reb D, 250 μL of 80 g/L β-1,3-GT SEQ IDNO: 548 stock solution, 250 μL of 10 g/L SuS SEQ ID NO: 1158 stocksolution and 500 μL of 2.6 g/L ADP stock solution in 400 g/L of sucrose.The buffer used was 50 mM pH 7.0 potassium phosphate with 3 mM MgSO₄.Enzyme and sucrose stock solution (400 g/L) was prepared in the buffer.The final compositions was 100 g/L Reb D, 200 g/L sucrose, 20 g/L SEQ IDNO: 548, 2.5 g/L SEQ ID NO: 1158, and 1.3 g/L (3 mM) of ADP. Theresulting thick slurry was stirred under air. The progress of thereaction was followed by taking aliquots and quenched with 1:1acetonitrile/water. After centrifuging at 4000 rpm/r.t./5 min, the clearhomogeneous supernatant was analyzed by HPLC using the instrument andparameters listed in Table 21.1 and Table 21.2. After 48 h, the reactionmixture consisted of >90% Reb M (Table 23.1).

TABLE 23.1 Conversion of Rebaudioside D to Rebaudioside M Over Time. t[h] % Reb M 0 0 1 16 2 28 4 40 8 52 24 79 32 88 48 94

Example 24 Transformation of Stevioside to Rebaudioside A, RebaudiosideA to Rebaudioside D, and Rebaudioside D to Rebaudioside M with AGT andACSuS

In this Example, experiments conducted to produce rebaudioside A fromstevioside, rebaudioside D from rebaudioside A, and rebaudioside M fromrebaudioside D are described. To a vial under air was added 250 μL of 20g/L β-1,3-GT SEQ ID NO: 1290 stock solution, 250 μL of 40 g/L β-1,2-GTSEQ ID NO: 954 stock solution, and 500 μL of 50 g/L Reb A 60, 2.6 g/LADP, 5 g/L β-1,3-GT SEQ ID NO: 548, and 1 g/L SuS SEQ ID NO: 1158 stocksolution, in 400 g/L of sucrose. The buffer used was 50 mM pH 7.0potassium phosphate with 3 mM MgSO₄. Enzyme and sucrose stock solution(400 g/L) was prepared in the buffer. The final compositions was 25 g/LReb A 60, 200 g/L sucrose, 5 g/L SEQ ID NO: 1290, 10 g/L SEQ ID NO: 954,2.5 g/L SEQ ID NO: 548, 0.5 g/L SEQ ID NO: 1158 and 1.3 g/L (3 mM) ofADP. The resulting clear homogenous solution was stirred under air (thereaction gradually turned cloudy with formation of precipitates). Theprogress of the reaction was followed by taking aliquots and quenchedwith 1:1 acetonitrile/water. After centrifuging at 4000 rpm/r.t./5 min,the clear homogeneous supernatant was analyzed by HPLC using theinstrument and parameters listed in Table 21.1 and Table 21.2. After 24h, <5% stevioside remained and formation of 9% of Reb M was observed(Table 24.1).

TABLE 24.1 Conversion of Stevioside and Rebaudioside A to GlucosylatedProducts Over Time. t [h] % stevioside % Reb A % Reb D % Reb I % Reb M 035 65 0 0 0 1 29 71 0 0 0 2 26 73 0 1 0 4 2 97 0 1 0 8 8 91 0 1 0 24 170 0 18 9

Example 25 Immobilization of AGT and/or ACSuS

In this Example, experiments conducted to immobilize an AGT (e.g., thevariants of SEQ ID NOS: 1290, 954, and/or 548) and/or an ACSuS variant(e.g., SEQ ID NO:1158) are described. To a vessel is added the enzymesolution, either alone or as a combination of enzymes (i.e., AGT and/orACSuS enzymes), and the solid support. The solid support is cationic,anionic, hydrophobic, hydrophilic with or without the presence ofcovalent bond forming functional groups such as thiol, alcohol, amines,olefin, alkyl halide and/or epoxide. The solid support is either adiscrete polymeric resin or amorphous (nano) clay or activated carbon.The magnetic particle is used when it is suitable for productisolation/enzyme recycling. The reaction is carried out in the presenceor absence of glutaraldehyde. The progress of enzyme uptake by the solidsupport is followed by Bradford assay. Alternatively, the solid supportis packed in a column and the enzyme solution is flowed through thecolumn, with recycling if necessary, until the desired degree of enzymecapture is reached. In some embodiments, all enzymes of interest areimmobilized on a same solid support in the same reaction vessel, whilein some alternative embodiments, the enzymes are immobilizedindividually in separate vessels or a combination thereof. In someembodiments, the immobilized enzyme is either isolated via filtration oris used immediately by adding buffer, sucrose, ADP and substrate to theimmobilization reaction mixture.

Example 26 Transformation of Stevioside to Rebaudioside A withImmobilized AGT and/or ACSuS and Recycling of Sugar Solution

In this Example, transformation of stevioside to rebaudioside A using animmobilized AGT (e.g., SEQ ID NO: 1290) and ACSuS (e.g., SEQ ID NO:1158), and sugar solution recycling are described. To a vessel is addedimmobilized β-1,3-GT (e.g., SEQ ID NO: 1290) and immobilized SuS (e.g.,SEQ ID NO: 1158). Alternatively, in some embodiments, one of the enzymesis used in the immobilized form and the other is used in solution form.After the addition of buffer, sucrose, ADP and substrate (i.e.,stevioside or Reb A 60), the reaction is monitored until the desiredconversion is reached. In some embodiments, the product and immobilizedenzyme are isolated by filtration. In some embodiments, immobilizedenzyme is further separated from the product via either centrifugation,particle size filtration or magnetic retrieval and is re-used. The sugarfiltrate is returned to the vessel for the next iteration.

Example 27 Transformation of Rebaudioside A to Rebaudioside D withImmobilized AGT and/or ACSuS and Recycling of Sugar Solution

In this Example, experiments conducted to produce rebaudioside D fromrebaudioside D with immobilized AGT and ACSuS, and sugar solutionrecycling are described. To a vessel is added immobilized β-1,2-GT(e.g., SEQ ID NO: 954) and immobilized SuS (e.g., SEQ ID NO: 1158).Alternatively, in some embodiments, one of the enzymes is used in theimmobilized form and the other is used in solution form. After theaddition of buffer, sucrose, ADP and substrate (stevioside or Reb A 60),the reaction is monitored until the desired conversion is reached. Insome embodiments, the product and immobilized enzyme is isolated byfiltration. In some embodiments, immobilized enzyme is further separatedfrom the product via either centrifugation, particle size filtration ormagnetic retrieval and is re-used. The sugar filtrate is returned to thevessel for the next iteration.

Example 28 Transformation of Rebaudioside D to Rebaudioside M withImmobilized AGT and/or ACSuS and Recycling of Sugar Solution

In this Example, experiments conducted to produce rebaudioside M fromrebaudioside D with immobilized AGT and ACSuS, and sugar solutionrecycling are described. To a vessel is added immobilized β-1,3-GT(e.g., SEQ ID NO: 548) and immobilized SuS (e.g., SEQ ID NO: 1158).Alternatively, in some embodiments, one of the enzymes is used in theimmobilized form and the other is used in solution form. After theaddition of buffer, sucrose, ADP and Reb D, the reaction is monitoreduntil the desired conversion is reached. In some embodiments, theproduct and immobilized enzyme are isolated by filtration. In someadditional embodiments, immobilized enzyme is further separated from theproduct via either centrifugation, particle size filtration or magneticretrieval and is re-used. The sugar filtrate is returned to the vesselfor the next iteration.

Example 29 Transformation of Stevioside to Rebaudioside A andRebaudioside A to Rebaudioside D with Immobilized AGT and ACSuS andRecycling of Sugar Solution

In this Example, experiments conducted to produce rebaudioside A fromstevioside, and rebaudioside D from rebaudioside A with immobilized AGTand ACSuS, and sugar solution recycling are described. To a vessel isadded immobilized β-1,3-GT (e.g., SEQ ID NO: 1290) and β-1,2-GT (e.g.,SEQ ID NO: 954) and immobilized SuS (e.g., SEQ ID NO: 1158).Alternatively, one or two of these enzymes is used in the immobilizedform and the others are used in solution form. After the addition ofbuffer, sucrose, ADP and substrate (stevioside or Reb A 60), thereaction is monitored until the desired conversion is reached. In someembodiments, the product and immobilized enzyme are isolated byfiltration. In some embodiments, immobilized enzyme is further separatedfrom the product via either centrifugation, particle size filtration ormagnetic retrieval and is re-used. The sugar filtrate is returned to thevessel for the next iteration.

Example 30 Transformation of Stevioside to Rebaudioside A, RebaudiosideA to Rebaudioside D and Rebaudioside D to Rebaudioside M withImmobilized AGT and ACSuS and Recycling of Sugar Solution

To a vessel is added immobilized β-1,3-GT (e.g., SEQ ID NO: 1290),β-1,2-GT (e.g., SEQ ID NO: 954), β-1,3-GT (e.g., SEQ ID NO: 548), andimmobilized SuS (e.g., SEQ ID NO: 1158). Alternatively, in someembodiments, one, two or three of these enzymes is/are used in theimmobilized form and the others are used in solution form. After theaddition of buffer, sucrose, ADP and substrate (stevioside or Reb A 60),the reaction is monitored until the desired conversion is reached. Insome embodiments, the product and immobilized enzyme are isolated byfiltration. In some embodiments, immobilized enzyme is further separatedfrom the product via either centrifugation, particle size filtration ormagnetic retrieval and is re-used. The sugar filtrate is returned to thevessel for the next iteration.

Example 31 Sucrose Synthase Variants of SEQ ID NO: 1222

Directed evolution of the SuS encoded by SEQ ID NO: 1222 was continuedby constructing libraries of variant genes in which mutations associatedwith improved activity in earlier rounds of evolution were recombined.These libraries were then plated, grown, and screened using thehigh-throughput (HTP) assay described below, to provide a fourth round(“Round 4”) of 49 engineered SuS variant polypeptides with activitytoward the generation of ADP-glucose.

HTP Coupled Assay for Glucose Transfer from Sucrose to ADP toRebaudioside D

Libraries were screened using the following HTP enzyme-coupled assay.Pelleted E. coli cultures were lysed with 250 μL of Tris-HCl, pH 7.5,with 1 mM magnesium sulfate and 0.5 mg/mL lysozyme and polymyxin Bsulfate (PMBS) and cleared by centrifugation. Lysate was diluted 20×into Tris-HCl, pH 7.5. Then, 10 μL diluted SuS lysate and 2 g/L GT SEQID NO: 696 (Rd8BB) were combined in 100 μL reaction volume withsubstrate loading of −1 mM rebaudioside D and co-substrate loadings of 1Mm ADP (Sigma, >95%) and 10 mM sucrose (Sigma). The following reactionconditions were used: 50 mM potassium phosphate buffer, pH7, 3 mM MgCl₂,50° C., in a Thermotron® titre-plate shaker with 300 RPM shaking for 2h.The reaction described above was quenched by adding 10 μL assay mixtureto 90 μL acetonitrile with 0.2% formic acid, and precipitated bycentrifugation. The supernatant was diluted 10× in water and steviolglycoside products were detected by RapidFire SPE-MS/MS, with theinstrument and parameters described in Table 31.1.

TABLE 31.1 RapidFire SPE-MS/MS Conditions for Steviol GlycosideDetection. Agilent RapidFire Conditions Buffer A 0.1% formic acid inLC/MS grade water; 1.5 mL/min flow rate Buffer B 0.1% formic acid inLC/MS grade methanol; 0.8 mL/min flow rate Aqueous wash Water Organicwash Acetonitrile SPE cartridge Agilent RapidFire cartridge A (C4) RFstate 1 600 ms RF state 2 2500 ms RF state 3 0 RF state 4 5000 ms RFstate 5 1000 ms Agilent Jet Stream Source Parameters Drying gastemperature 300° C. Drying gas flow 10 L/min Nebulizer pressure 45 psiSheath gas temperature 350° C. Sheath gas flow 11 L/min Capillaryvoltage +3500 V Nozzle voltage +2000 V Agilent 6470 Triple QuadrupoleMRM Parameters Compound Q1 Q3 Dwell Fragmentor CE CAV Stevioside 827.4665.3 50 150 50 5 RebA 989.5 827.5 50 350 60 5 RebD or RebI 1151.7 827.550 350 55 5 RebM 1313.7 827.5 50 350 70 5

After analysis, 49 engineered SuS variant polypeptides that showedimproved activity coupled with a GT on rebaudioside D were identified.The resulting engineered polypeptides are listed in Table 31.2.Shake-flask scale cultures were grown for protein characterization asdescribed in below for variants with the amino acid mutations shown inTable 31.3.

TABLE 31.2 SUS Round 4 Variants and RebM Levels SEQ ID NO: Amino AcidDifferences Increased (nt/aa) (Relative to SEQ ID NO: 1222) RebM^(a)1295/1296 H47L/I372V/A635S/Q776E + 1297/1298H47L/R93V/L98V/P358E/R583Q/A635S + 1299/1300V68A/R93V/R154H/P358E/I372V/R440P/ + Q776E 1301/1302 I372V/W375Y/Q776E +1303/1304 H47L/L98V/R129E/P358E/I372V/W375Y/ + H438Q/E534H 1305/1306R154H/E534H/A635S/K724H + 1307/1308 R93V/R154H/S248A/K724H + 1309/1310H47L/R93V/P358E/I372V/W375Y/Q776E + 1311/1312 V68A/R136S/K724H +1313/1314 H47L/P358E/A635S/Q776E + 1315/1316 L98V/E534H/R583Q/A635S +1317/1318 R93V/L98V/T125E/R154H/S250D/R440P + 1319/1320R93V/L98V/R154H/A635S/Q776E +++ 1321/1322R129E/R154H/S248A/S250D/P358E/W375Y/ +++ E534H/A635S 1323/1324R93V/T125E/R154H/R440P/E534H ++ 1325/1326 R93V/L98V/E534H + 1327/1328R93V/L98V/R154H/S250D/P358E/W375Y/ ++ E534H 1329/1330H47L/L98V/I372V/W375Y ++ 1331/1332 H47L/R93V/L98V/R154H/I372V/W375Y/ +++Q776E 1333/1334 H47L/R93V/R129E/R136S/R154H/S250D/ +I372V/E534H/A635S/K724H 1335/1336 R93V/R129E/S250D/P358E/I372V/W375Y/ +R583Q 1337/1338 V68A/W375Y/R440P/E534H/K724H/Q776E + 1339/1340H47L/T125E/R154H +++ 1341/1342 R93V/L98V/T125E/R154H/S248A ++ 1343/1344V68A/R154H/E534H/A635S ++ 1345/1346 H47L/R93V/R129E/R136S/W375Y/E534H/++ R583Q 1347/1348 V68A/R129E/R583Q/K724H ++ 1349/1350H47L/R93V/L98V/R136S/R154H/E772G/ +++ Q776E 1351/1352H47L/R136S/R583Q/Q776E ++ 1353/1354 R154H/S250D/P358E/W375Y/R583Q +++1355/1356 H47L/R129E/S248A/S250D/I372V/W375Y/ + E534H/K724H 1357/1358W375Y/A635S + 1359/1360 H47L/P358E/R440P/K724H + 1361/1362R129E/R136S/W375Y + 1363/1364 H47L/V68A/R93V/L98V/P358E/R440P +1365/1366 L98V/R129E/W375Y/R583Q + 1367/1368 R93V/P358E/E534H/A635S +1369/1370 E534H/R583Q + 1371/1372 H47L/V68A/R154H/I372V/W375Y +1373/1374 R129E/P358E/I372V/A635S ++ 1375/1376H47L/R129E/R136S/I372V/W375Y/E534H ++ 1377/1378R93V/L98V/R129E/R154H/S248A ++ 1379/1380 V68A/R129E/R440P +++ 1381/1382R154H/P358E/W375Y/E534H/Q776E +++ 1383/1384H47L/L98V/R129E/W375Y/E534H/A635S/ ++ K724H/Q776E 1385/1386V68A/R154H/P358E/W375Y ++ 1387/1388 V68A/R93V/L98V/R129E/P358E/W375Y/ ++K724H 1389/1390 R129E/R136S/R154H/S248A/S250D/I372V/ ++ W375Y/E534H1391/1392 H47L/R93V/P358E/I372V/W375Y/R440P/ +++ K724H ^(a)Levels ofincreased production were determined relative to the referencepolypeptide of SEQ ID NO: 1222, and are defined as follows: “+” =production at least 2-fold, but less than 3.05-fold; “++” = at least3.05-fold, but less than 3.29-fold; and “+++” = at least 3.29-foldincreased production, as compared to the reference polypeptide.

Production of Shake Flask Powders (SFP)

A shake-flask procedure was used to generate the glycosyltransferasepolypeptide shake flask powders (SFP) for characterization assays usedin the biocatalytic processes described herein. Shake flask powder (SFP)preparation of enzymes provides amore purified preparation (e.g., upto >30% of total protein) of the enzyme, as compared to the cell lysateused in HTP assays, and also allows for the use of more concentratedenzyme solutions. A single colony of E. coli containing a plasmidencoding an engineered polypeptide of interest was inoculated into 5 mLLuria Bertani broth containing 30 μg/ml chloramphenicol and 1% glucose.Cells were grown overnight (at least 16 hours) in an incubator at 30° C.with shaking at 250 rpm. The culture was diluted into 250 mL TerrificBroth (12 g/L bacto-tryptone, 24 g/L yeast extract, 4 mL/L glycerol, 65mM potassium phosphate, pH 7.0, 1 mM MgS0₄) containing 30 μg/ml CAM, ina 1 L flask to an optical density of 600 nm (OD600) of 0.2, and allowedto grow at 30° C.

Shake Flask Powder Characterization Assay and Analysis for GlucosylTransfer from Sucrose to Rebaudioside D

An experiment was performed to characterize the activity of theengineered round 4 SUS variants on sucrose and ADP to facilitate theformation of rebaudioside M from rebaudioside D. Shake flask powder(SFP) was added to a 100 μL total reaction volume at 0.125 g/Lconcentration containing 50 mM potassium phosphate buffer, pH 7, 3 mMmagnesium chloride, 1 g/L rebaudioside D, 10 mM sucrose, 1 mM ADP, and 2g/L GT SEQ ID NO: 734. The reaction was performed at 50° C. in aThermotron® titre-plate shaker with 300 RPM shaking for 1 h. Thereaction was quenched by adding 10 μL of the reaction mixture to 90 μLacetonitrile with 0.2% formic acid, and precipitated by centrifugation.The supernatant was diluted 10× in water and analyzed for steviolglycosides by RapidFire SPE-MS/MS, with the instrument and parametersdescribed in Table 31.1. At least 3 of the variants had higheractivities than SEQ ID NO: 1222. The levels of rebaudioside M producedfrom rebaudioside D by the variants relative to SEQ ID NO: 1222 arelisted in Table 31.3. The variant with mutations H47L, R93V, P358E,I372V, W375Y, R440P, and K724H (SEQ ID NO: 1392), relative to SEQ ID NO:1222, had the highest activity. Therefore, the encoding polynucleotide(SEQ ID NO: 1391) was selected for further directed evolution for thecatalysis of the recycling reaction transferring a glucose from sucroseto ADP.

TABLE 31.3 SUS Round 4 SFP Variants and RebM Levels SEQ ID NO: AminoAcid Differences Increased (nt/aa) (Relative to SEQ ID NO: 1222)RebM^(a) 1391/1392 H47L/R93V/P358E/I372V/W375Y/R440P/ + K724H 1349/1350H47L/R93V/L98V/R136S/R154H/E772G/ + Q776E 1381/1382R154H/P358E/W375Y/E534H/Q776E + 1365/1366 L98V/R129E/W375Y/R583Q +1379/1380 V68A/R129E/R440P + 1391/1392H47L/R93V/P358E/I372V/W375Y/R440P/ + K724H ^(a)Levels of increasedproduction were determined relative to the reference polypeptide of SEQID NO: 1222, and defined as follows: “+” = production at least1.04-fold, but less than 1.2-fold greater productivity, as compared tothe reference polypeptide.

Example 32 Sucrose Synthase Variants of SEQ ID NO: 1392

Directed evolution of the SuS encoded by SEQ ID NO: 1391 was continuedby constructing libraries of variant genes in which mutations associatedwith improved activity in earlier rounds of evolution were recombined.These libraries were then plated, grown, and screened using thehigh-throughput (HTP) assay described below, to provide a fifth round(“Round 5”) of 86 engineered SuS variant polypeptides with activitytoward the generation of ADP-glucose.

HTP Coupled Assay for Glucose Transfer from Sucrose to ADP toRebaudioside D

Libraries were screened using the following HTP enzyme-coupled assay.Pelleted E. coli cultures were lysed with 400 μL of Tris-HCl, pH 7.5with 1 mM magnesium sulfate and 0.5 mg/mL lysozyme and polymyxin Bsulfate (PMBS) and cleared by centrifugation. Lysate was diluted 25-60×into Tris-HCl, pH 7.5. 10 μL diluted SuS lysate and 2 g/L GT SEQ ID NO:4684 (Rd9BB) in 100 μL reaction volume with substrate loading of ˜1 mMrebaudioside D and co-substrate loadings of 1 mM ADP (Sigma, >95%) and10 mM sucrose (Sigma). The following reaction conditions were used: 50mM potassium phosphate buffer, pH 7, 3 mM MgCl₂, 50° C. in a Thermotron®titre-plate shaker with 300 RPM shaking for 1 h. The reaction describedabove was quenched by adding 10 μL assay to 90 μL acetonitrile with 0.2%formic acid, and precipitated by centrifugation. The supernatant wasdiluted 10× in water and steviol glycoside products were detected byRapidFire SPE-MS/MS, with the instrument and parameters described inTable 31.1.

After analysis, 86 engineered SuS variant polypeptides that showedimproved activity coupled with a GT on rebaudioside D were identified.The resulting engineered polypeptides are listed in Table 32.1.Shake-flask scale cultures were grown for protein characterization asdescribed in below for variants with the amino acid mutations shown inTable 32.2.

TABLE 32.1 SUS Round 5 Variants and RebM Levels SEQ ID NO: Amino AcidDifferences Increased (nt/aa) (Relative to SEQ ID NO: 1392) RebM^(a)1395/1396 G54D/A97V/A118N/N307E/G694N/L727E/ + A738E 1397/1398A97V/A118N/H442N/G694N/L727E/A738E + 1399/1400 Y17D/F684H + 1401/1402Y17D/Y434H/F684H + 1403/1404 Y17D/Y357K/Y434H/F519T/F684H ++ 1405/1406Y17D/Y357K/P364R/Y434H/F519T/F684H ++ 1407/1408 R550I + 1409/1410V68A/L98V/R129E/R136S ++ 1411/1412 R136S/G603D ++ 1413/1414L98V/R136S/R154H/A635S ++ 1415/1416 V68A/R136S/E534H/A635S ++ 1417/1418V68A/R154H ++ 1419/1420 V68A/R129E/R136S/R154H ++ 1421/1422L98V/R129E/R136S/R154H/A635S +++ 1423/1424 V68A/L98V/R154H/E534H/A635S++ 1425/1426 L98V/R154H + 1427/1428 R136S/E534H/A635S + 1429/1430L98V/R154H/E534H ++ 1431/1432 V68A/L98V/R136S/R154H/A635S ++ 1433/1434R129E/A635S + 1435/1436 R129E/R154H/A635S +++ 1437/1438V68A/L98V/R154H/E534H + 1439/1440 V68A/R129E/R136S + 1441/1442V68A/R129E/R136S/R154H/I464F/A635S +++ 1443/1444 L98V/R129E/R154H +++1445/1446 R154H/A635S ++ 1447/1448 R129E/R154H/E534H ++ 1449/1450R154H + 1451/1452 L98V/R136S/A635S ++ 1453/1454V68A/R136S/R154H/E534H/A635S +++ 1455/1456 V68A/L98V/R129E/R154H/A635S+++ 1457/1458 V68A/R129E/R154H ++ 1459/1460 V68A/R154H/A635S ++1461/1462 V68A/L98V/R154H + 1463/1464 L98V/R136S/E534H/A635S ++1465/1466 V68A/L98V/R136S/R154H/E534H/A635S +++ 1467/1468L98V/R129E/E534H/A635S ++ 1469/1470 V68A/L98V/R129E/R136S/R154H ++1471/1472 R129E/R136S/E534H + 1473/1474 V68A/R129E/R136S/E534H/A635S +++1475/1476 V68A/R129E/R154H/D765H +++ 1477/1478L98V/R129E/R154H/E534H/A635S +++ 1479/1480 V68A/R136S/A635S ++ 1481/1482V68A/L98V/R129E/R136S/R154H/E534H ++ 1483/1484 R129E/R136S/A635S +++1485/1486 R136S/R154H/A635S +++ 1487/1488 L98V/R129E/R136S/E534H +1489/1490 L98V/R129E/R136S/R154H ++ 1491/1492 R136S/A635S ++ 1493/1494R132C/R136S/R154H/E534H/A635S ++ 1495/1496 L98V/R129E/R136S/A635S +++1497/1498 R129E/R136S/R154H/A635S +++ 1499/1500 R129E/R136S + 1501/1502V68A/R154H/E534H/A635S +++ 1503/1504 V68A/L98V/R129E/R154H/E534H +++1505/1506 F160W + 1507/1508 S161Q + 1509/1510 F160M + 1511/1512 A635E +1513/1514 A253G + 1515/1516 F160E + 1517/1518 F160A + 1519/1520 Q381S +1521/1522 R550M + 1523/1524 F519A + 1525/1526 P785D + 1527/1528 R167E +1529/1530 L563V + 1531/1532 A635D ++ 1533/1534 P285A + 1535/1536 R550Q +1537/1538 F519L + 1539/1540 F519T + 1541/1542 A635R + 1543/1544 F519G +1545/1546 G157A ++ 1547/1548 A122E + 1549/1550 S564A +++ 1551/1552A122D + 1553/1554 R550S + 1555/1556 A253T + 1557/1558 F160S + 1559/1560F160N + 1561/1562 F519S + 1563/1564 G157F + 1565/1566 A253V + ^(a)Levelsof increased production were determined relative to the referencepolypeptide of SEQ ID NO: 1392, and are defined as follows: “+” =production at least 1.2-fold, but less than 1.54-fold; “++” = at least1.54-fold, but less than 1.73-fold; and “+++” production at least1.73-fold greater than that of the reference polypeptide.Shake Flask Powder Characterization Assay and Analysis for GlucosylTransfer from Sucrose to Rebaudioside D

An experiment was performed to characterize the activity of theengineered round 4 SUS variants on sucrose and ADP to facilitate theformation of rebaudioside M from rebaudioside D. Shake flask powder(SFP) was added to a 100 μL total reaction mixture volume at 0.05 g/Lconcentration containing 50 mM potassium phosphate buffer, pH 7, 3 mMmagnesium chloride, 1 g/L rebaudioside D, 10 mM sucrose, 1 mM ADP, and 2g/L GT SEQ ID NO: 4684. The reaction was performed at 50° C. in aThermotron® titre-plate shaker with 300 RPM shaking for 1-2 h. Thereaction was solubilized by diluting 2.5× into water, quenched by adding10 μL of the diluted reaction to 90 μL acetonitrile with 0.2% formicacid, and precipitated by centrifugation. The supernatant was diluted3.33× in water and analyzed for steviol glycosides by RapidFireSPE-MS/MS, with the instrument and parameters described in Table 31.1.All of the variants in Table 32.2 had higher activities than SEQ ID NO:1392. The levels of rebaudioside M produced from rebaudioside D by thevariants relative to SEQ ID NO: 1392 are listed in Table 32.2. Thevariant with mutations V68A, L98V, R129E, R154H, and A635S (SEQ ID NO:1455) relative to SEQ ID NO: 1392, was selected for further directedevolution for the catalysis of the recycling reaction transferring aglucose from sucrose to ADP.

TABLE 32.2 SUS Round 5 SFP Variants and RebM Levels SEQ ID NO: AminoAcid Differences Increased (nt/aa) SEQ ID NO: 1392) RebM^(a) 1393/1394R154Q/S161T + 1397/1398 A97V/A118N/H442N/G694N/L727E/A738E + 1405/1406Y17D/Y357K/P364R/Y434H/F519T/F684H +++ 1407/1408 R550I + 1455/1456V68A/L98V/R129E/R154H/A635S ++ 1477/1478 L98V/R129E/R154H/E534H/A635S +1497/1498 R129E/R136S/R154H/A635S ++ ^(a)Levels of increased productionwere determined relative to the reference polypeptide of SEQ ID NO:1392, and are defined as follows: “+” = production at least that of thereference polypeptide, but less than 1.12-fold; “++” = production atleast 1.12-fold, but less than 1.20-fold; and “+++” = production atleast 1.20-fold greater, as compared to the reference polypeptide.

Example 33 Sucrose Synthase Variants of SEQ ID NO: 1456

Directed evolution of the SuS encoded by SEQ ID NO: 1455 was continuedby constructing libraries of variant genes in which mutations associatedwith improved activity in earlier rounds of evolution were recombined.These libraries were then plated, grown, and screened using thehigh-throughput (HTP) assay described below, to provide a sixth round(“Round 6”) of 16 engineered SuS variant polypeptides with activitytoward the generation of ADP-glucose.

HTP Coupled Assay for Glucose Transfer from Sucrose to ADP toRebaudioside A

Libraries were screened using the following HTP enzyme-coupled assay.Pelleted E. coli cultures were lysed with 400 μL of Tris-HCl, pH 7.5,with 1 mM magnesium sulfate and 0.5 mg/mL lysozyme and polymyxin Bsulfate (PMBS) and cleared by centrifugation. Lysate was diluted 30×into Tris-HCl, pH 7.5. Then, 10 μL diluted SuS lysate and 1 g/L GT SEQID NO: 2814 (Rd8BB) were combined in 100 μL reaction volume withsubstrate loading of 4.5 mM rebaudioside A 97 and co-substrate loadingsof 0.25 mM ADP (Sigma, >95%) and 10 mM sucrose (Sigma). The followingreaction conditions were used: 50 mM potassium phosphate buffer, pH 6.5,50° C., in a Thermotron® titre-plate shaker with 300 RPM shaking for 1h. The reaction described above was solubilized by adding 10 μL assaymixture to 90 μL water, quenched by adding 10 μL solubilized assaymixture to 90 μL acetonitrile with 0.2% formic acid, and precipitated bycentrifugation. The supernatant was diluted 7.3× in water, and steviolglycoside products were detected by RapidFire SPE-MS/MS, with theinstrument and parameters described in Table 31.1.

After analysis, 16 engineered SuS variant polypeptides that showedimproved activity coupled with a GT on rebaudioside A were identified.The resulting engineered polypeptides are listed in Table 33.1.Shake-flask scale cultures were grown for protein characterization asdescribed in Example 31, for variants with the amino acid mutationsshown in Table 33.2.

TABLE 33.1 SUS Round 6 Variants and RebD Levels SEQ ID NO: Amino AcidDifferences Increased (nt/aa) (Relative to SEQ ID NO: 1456) RebD^(a)1567/1568 Y17D/G54D/A524T/R550I/L727E + 1569/1570Y17D/Y434H/A524T/F684H + 1571/1572 Y17D/A738E + 1573/1574Y17D/G54D/A97V/R136P/E329Q/R550I/ + F684H/A738E 1575/1576G54D/A97V/Y434H/A524T/R550I/F684H/ + L727E 1577/1578Y17D/S161T/Y434H/A524T/R766H ++ 1579/1580E329Q/R550I/F684H/L727E/A738E + 1581/1582Y17D/G54D/S161T/F519T/L727E/A738E ++ 1583/1584 Y17D/Y434H/A738E +++1585/1586 G54D/A97V/S161T/Y434H/H442N +++ 1587/1588G54D/R136P/H442N/R550I ++ 1589/1590 Y17D/G54D/A97V/E329Q/A524T/F684H ++1591/1592 G54D/Y434H/A524T/A738E ++ 1593/1594Y17D/H442N/A524T/R550I/F684H/E721K + 1595/1596 Y17D/L727E +++ 1597/1598A97V/R136P/F519T/R550I/L727E/A738E + ^(a)Levels of increased productionwere determined relative to the reference polypeptide of SEQ ID NO:1456, and defined as follows: “+” = production at least 1.4-fold, butless than 1.5-fold; “++” = greater than 1.5-fold, but less than1.54-fold; and “+++” = greater than 1.54-fold increased production,relative to the reference polypeptide.Shake Flask Powder Characterization Assay and Analysis for GlucosylTransfer from Sucrose to Rebaudioside A

An experiment was performed to characterize the activity of theengineered round 6 SUS variants on sucrose and ADP to facilitate theformation of rebaudioside D from rebaudioside A. Shake flask powder(SFP) was added to a 100 μL total reaction volume at 0.01 g/Lconcentration containing 50 mM potassium phosphate buffer, pH 6.5, 4.5mM rebaudioside A 97, 30 mM sucrose, 0.25 mM ADP, and 1 g/L GT SEQ IDNO: 2814. The reaction was performed at 50° C. in a Thermotron®titre-plate shaker with 300 RPM shaking for 1-2 h. The reaction wassolubilized by diluting 10× into water, quenched by adding 10 μL of thediluted reaction to 90 μL acetonitrile with 0.2% formic acid, andprecipitated by centrifugation. The supernatant was diluted 4.4× inwater and analyzed for steviol glycosides by RapidFire SPE-MS/MS, withthe instrument and parameters described in Table 31.1. At least 4 of thevariants had higher activities than SEQ ID NO: 1456. The levels ofrebaudioside D produced from rebaudioside A by the variants relative toSEQ ID NO: 1456 are listed in Table 33.2. The variant with mutationsY17D, G54D, S161T, F519T, L727E, and A738E (SEQ ID NO: 1582) relative toSEQ ID NO: 1456, was selected for further directed evolution for thecatalysis of the recycling reaction transferring a glucose from sucroseto ADP.

TABLE 33.2 SUS Round 6 SFP Variants and RebD Levels SEQ ID NO: AminoAcid Differences Increased (nt/aa) (Relative to SEQ ID NO: 1456)RebD^(a) 1577/1578 Y17D/S161T/Y434H/A524T/R766H + 1581/1582Y17D/G54D/S161T/F519T/L727E/A738E + 1583/1584 Y17D/Y434H/A738E +1595/1596 Y17D/L727E + ^(a)Levels of increased production weredetermined relative to the reference polypeptide of SEQ ID NO: 1456, anddefined as follows: “+” = production between 1.15- and 1.36-fold greaterthan that of the reference polypeptide.

Example 34 Sucrose Synthase Variants of SEQ ID NO: 1582

Directed evolution of the sucrose synthase encoded by SEQ ID NO: 1581was continued by constructing libraries of variant genes in whichmutations associated with improved activity in earlier rounds ofevolution were recombined. These libraries were then plated, grown, andscreened using the high-throughput (HTP) assay described below, toprovide a seventh round (“Round 7”) of 87 engineered SuS variantpolypeptides with activity toward the generation of ADP-glucose.

HTP Coupled Assay for Glucose Transfer from Sucrose to ADP toRebaudioside A

Combinatorial libraries were screened using the following HTPenzyme-coupled assay. Pelleted E. coli cultures were lysed with 400 μLof Tris-HCl, pH 7.5 with 1 mM magnesium sulfate and 0.5 mg/mL lysozymeand polymyxin B sulfate (PMBS), and cleared by centrifugation. Lysatewas diluted ˜90× into Tris-HCl, pH7.5. Then, 10 μL diluted SuS lysateand 1 μL GT SEQ ID NO: 2884 (Rd9BB) were combined in 100 μL reactionvolume with substrate loading of 4.5-7.5 mM rebaudioside A97 andco-substrate loadings of 0.2-0.25 mM ADP (Sigma, >95%) and 30 mM sucrose(Sigma). The following reaction conditions were used: 50 mM potassiumphosphate buffer, pH 6, 55° C. in a Thermotron® titre-plate shaker with300 RPM shaking for 2h. The reaction described above was solubilized byadding 10 μL assay to 90-190 μL water, quenched by adding 10 μLsolubilized assay to 90 μL acetonitrile with 0.2% formic acid, andprecipitated by centrifugation. The supernatant was diluted 4.4-6.7× inwater and steviol glycoside products were detected by RapidFireSPE-MS/MS, with the instrument and parameters described in Table 31.1.

After analysis, 87 engineered SuS variant polypeptides that showedimproved activity coupled with a GT on rebaudioside A were identified.The resulting engineered polypeptides are listed in Table 34.1.Shake-flask scale cultures were grown for protein characterization asdescribed in Example 31 for variants with the amino acid mutations shownin Table 34.2.

TABLE 34.1 SUS Round 7 Variants and RebD Levels SEQ ID NO: Amino AcidDifferences Increased (nt/aa) (Relative to SEQ ID NO: 1582) RebD^(a)1599/1600 L562I ++ 1601/1602 V270L/F452Y/P517A/I700F/L750M + 1603/1604P517A/L562I/L750M + 1605/1606 V270L/I367V/F452Y/P517A/E613Q/I700F/ ++L750M 1607/1608 V270L/R570H + 1609/1610 P517A + 1611/1612V270L/I322V/P517A + 1613/1614 L562I + 1615/1616 P517A/T640N + 1617/1618R71Q/R222L/R236S ++ 1619/1620 R14D/R137K/R356H/R745L + 1621/1622R14D/R570H/R745L +++ 1623/1624 R236S ++ 1625/1626 R356H/R570H +++1627/1628 R71Q/R158T/R222L/R356H + 1629/1630 R14D/R570H + 1631/1632R137K/R319K/R570H + 1633/1634 R71Q/R319K/R356H/R606S + 1635/1636S26E/A164E/A165E/A213E/R586E +++ 1637/1638 G117E/A608E ++ 1639/1640A213E/H365E/P517E/A707E ++ 1641/1642 G117E/R158E/A213E/G332E/A608E +1643/1644 S26E/G117E/H365E + 1645/1646 G117E/A164E/A707E + 1647/1648A165E/Q311E ++ 1649/1650 G117E/Q311E/G332E ++ 1651/1652G117E/A213E/H365E/P517E + 1653/1654 A253G/T519L + 1655/1656 T519L/L563V++ 1657/1658 A253T/T519L + 1659/1660 A253V/T519G ++ 1661/1662A253V/T519L + 1663/1664 A253T/T519L/L563V + 1665/1666 A253T/T519L/S635E++ 1667/1668 A253T/L563V/S635R ++ 1669/1670 G157A/A253T/T519L ++1671/1672 A253T/T519L/S635D +++ 1673/1674 A122E/R550Q + 1675/1676A122D/R167E + 1677/1678 F160W/L282M/Q381S/R550M + 1679/1680A122E/F1601/R167E/L282M/Q381H/R550Q/ ++ L636Q 1681/1682A122D/F160M/L282M/R550S + 1683/1684 F160M/T161Q/L282M/R550Q ++ 1685/1686A122D/R167E + 1687/1688 L282M/R550Q ++ 1689/1690 F160W/T161Q/L282M/R550M+++ 1691/1692 F160M/L282M/R550M/L636Q +++ 1693/1694A122D/L282M/Q381S/R550M +++ 1695/1696 A122D/R550M +++ 1697/1698 A122D+++ 1699/1700 A122E/L282M/R550Q +++ 1701/1702 T161Q/L282M/R550Q/L636Q+++ 1703/1704 A122E/F160M/T161Q/L282M/Q381S/R550M/ +++ L636Q 1705/1706R167E/R550Q ++ 1707/1708 A122D/L282M/R550Q + 1709/1710R167E/L282M/L636Q + 1711/1712 L282M/Q381S/R550S +++ 1713/1714Q381S/R550S + 1715/1716 T161Q/Q381S/R550Q + 1717/1718 A122E/R167E/R550M+++ 1719/1720 R550Q ++ 1721/1722 F160M/T161Q/L282M/Q381S/R550M ++1723/1724 A122E/F160W/Q381S/R550M/L636Q + 1725/1726F160M/L282M/Q381S/R550M ++ 1727/1728 A122D/F160W/T161Q/L282M/R550Q +1729/1730 F160M/T161Q +++ 1731/1732 Q381S/R550Q + 1733/1734A122E/F160W/Q381S/R550Q ++ 1735/1736 A122D/F160W/T161Q/R167E/R550M ++1737/1738 F160W/T161Q/L282M/R550Q +++ 1739/1740A122E/F160W/T161Q/R167E/R550S ++ 1741/1742 A122D/F160W/R550M + 1743/1744F160W/T161Q/R550Q/L636Q/A735V + 1745/1746 F160W/R167E/L282M/Q381S/L636Q++ 1747/1748 A122E/F160M/R550Q/L636Q + 1749/1750A122E/F160W/T161Q/L282M/Q381S/R550S + 1751/1752A122E/F160W/L282M/Q381S + 1753/1754 A122D/F160W/L282M/Q381S/R550M +1755/1756 A122E/F160W/T161Q/R550M/L636Q + 1757/1758 A122E/T161Q/R550Q +1759/1760 A122E/L282M/R550M/L636Q ++ 1761/1762 A122E/Q381S/E706K ++1763/1764 F160W/Q381S/R550Q/L636Q/A681V +++ 1765/1766 R550M/L636Q +1767/1768 A122E/L282M/R550S + 1769/1770 A122D/F160W/R550Q/L636Q +1771/1772 F160M/L282M + ^(a)Levels of increased production weredetermined relative to the reference polypeptide of SEQ ID NO: 1582, andare defined as follows: “+” = production at least that of the referencepolypeptide, but less than 1.42-fold; “++” = at least 1.42-fold, butless than 1.60-fold; and “+++” = at least 1.60-fold increasedproduction, relative to the reference polypeptide.Shake Flask Powder Characterization Assay and Analysis for GlucosylTransfer from Sucrose to Rebaudioside A

An experiment was performed to characterize the activity of theengineered round 7 SUS variants on sucrose and ADP to facilitate theformation of rebaudioside D from rebaudioside A. Shake flask powder(SFP) was added to a 100 μL total reaction volume at 0.02 μLconcentration containing 50 mM potassium phosphate buffer, pH6, 7.5 mMrebaudioside A97, 30 mM sucrose, 0.2 mM ADP, and 1 g/L GT SEQ ID NO:2884. The reaction was performed at 50° C. in a Thermotron® titre-plateshaker with 300 RPM shaking for 1h. The reaction was solubilized bydiluting 20× into water, quenched by adding 10 μL of the dilutedreaction to 90 μL acetonitrile with 0.2% formic acid, and precipitatedby centrifugation. The supernatant was diluted 4.4× in water andanalyzed for steviol glycosides by RapidFire SPE-MS/MS, with theinstrument and parameters described in Table 31.1. At least 3 of thevariants had higher activities than SEQ ID NO: 1582. The levels ofrebaudioside D produced from rebaudioside Aby the variants relative toSEQ ID NO: 1582 are listed in Table 34.2. The variant with mutationsF160W, Q381S, R550Q, L636Q, and A681V (SEQ ID NO:1764), relative to SEQID NO: 1582, was selected for further directed evolution for thecatalysis of the recycling reaction transferring a glucose from sucroseto ADP.

TABLE 34.2 SUS Round 7 SFP Variants and RebD Levels SEQ ID NO: AminoAcid Differences Increased (nt/aa) (Relative to SEQ ID NO: 1582)RebD^(a) 1599/1600 L562I + 1615/1616 P517A/T640N + 1763/1764F160W/Q381S/R550Q/L636Q/A681V + ^(a)Levels of increased production weredetermined relative to the reference polypeptide of SEQ ID NO: 1582, andare defined as follows: “+” = production between 1.1- and 1.25-foldgreater activity, as compared to the reference polypeptide.

Example 35 Sucrose Synthase Variants of SEQ ID NO: 1764

Directed evolution of the sucrose synthase encoded by SEQ ID NO: 1763was continued by constructing libraries of variant genes in whichmutations associated with improved activity in earlier rounds ofevolution were recombined and in which certain structural features weresubjected to saturation mutagenesis. These libraries were then plated,grown, and screened using the high-throughput (HTP) assay describedbelow, to provide an eighth and ninth round (“Round 8” and “Round 9”) of24 engineered SuS variant polypeptides with activity toward thegeneration of ADP-glucose.

HTP Coupled Assay for Glucose Transfer from Sucrose to ADP toRebaudioside A for Round 8

Combinatorial libraries were screened using the following HTPenzyme-coupled assay. Pelleted E. coli cultures were lysed with 400 μLof Tris-HCl, pH 7.5 with 1 mM magnesium sulfate and 0.5 mg/mL lysozymeand polymyxin B sulfate (PMBS), and cleared by centrifugation. Lysatewas diluted 15× into Tris-HCl, pH 7.5. Then, 10 μL diluted SuS lysateand 1 g/L GT SEQ ID NO: 2884 (Rd9BB) were combined in 100 μL reactionvolume with substrate loading of 15 mM rebaudioside A 97 andco-substrate loadings of 0.2 mM ADP (Sigma, >93%) and 45 mM sucrose(cane sugar). The following reaction conditions were used: 50 mMpotassium phosphate buffer, pH 6, 55° C. in a Thermotron® titre-plateshaker with 300 RPM shaking for 2 h. The reaction described above wassolubilized by adding 10 μL assay to 390 μL water, quenched by adding 20μL solubilized assay to 180 μL acetonitrile with 0.2% formic acid, andprecipitated by centrifugation. The supernatant was diluted 3.3× inwater and steviol glycoside products were detected by RapidFireSPE-MS/MS, with the instrument and parameters described in Table 31.1.No substantially improved combinatorial variants were identifiedrelative to SEQ ID NO: 1764, so another set of combinatorial andsaturation mutagenesis libraries were generated using the same backbone,and this set was called “Round 9”.

HTP Coupled Assay for Glucose Transfer from Sucrose to ADP toRebaudioside A for Round 9

Combinatorial libraries were screened using the following HTPenzyme-coupled assay. Pelleted E. coli cultures were lysed with 400 μLof Tris-HCl, pH 7.5 with 1 mM magnesium sulfate and 0.5 mg/mL lysozymeand polymyxin B sulfate (PMBS), and cleared by centrifugation. Undilutedlysate (saturation mutagenesis library) or 32× (Round 9 combinatoriallibraries) were added to Tris-HCl, pH 7.5. Round 9 library lysates werepre-incubated at 62° C. for 1 h (combinatorial libraries) or 3.75 h(saturation mutagenesis library) to thermally challenge the enzymes.Then, 10 μL diluted SuS lysate and 1 g/L GT SEQ ID NO: 3244 (Rd2B) werecombined in 100 μL reaction volume with substrate loading of 15 mMrebaudioside A97, co-substrate loadings of 0.2 mM ADP (Sigma, >95%), 45mM sucrose (cane sugar), and 9 mM fructose (Sigma). The followingreaction conditions were used: 50 mM potassium phosphate buffer, pH 6,60° C. in a Thermotron® titre-plate shaker with 300 RPM shaking for2-3h. The reaction described above was solubilized by adding 10 μL assayto 390 μL water, quenched by adding 20 μL solubilized assay to 180 μLacetonitrile with 0.2% formic acid, and precipitated by centrifugation.The supernatant was diluted 3.3× in water and steviol glycoside productswere detected by RapidFire SPE-MS/MS, with the instrument and parametersdescribed in Table 31.1. After analysis, 24 engineered SuS variantpolypeptides that showed improved activity coupled with a GT onrebaudioside A were identified. The resulting engineered polypeptidesare listed in Table 35.1. Shake-flask scale cultures were grown forprotein characterization as described in Example 31 for variants withthe amino acid mutations shown in Table 35.2.

TABLE 35.1 SUS Round 9 Variants and RebD Levels SEQ ID NO: Amino AcidDifferences Increased (nt/aa) (Relative to SEQ ID NO: 1764) RebD^(a)1773/1774 E536L +++ 1775/1776 H705P ++ 1777/1778 L580M + 1779/1780 H699F+++ 1781/1782 N347R/S532Y +++ 1783/1784 H705M + 1785/1786 A548P ++1787/1788 V681A +++ 1789/1790 L4071/R570H/V681A + 1791/1792 V270L/V681A++ 1793/1794 G117E/A122D/V270L/L540M/V681A + 1795/1796 L407T/V681A ++1797/1798 E536L/H705M + 1799/1800 A548P/L580M + 1801/1802N347R/E536L/A548P/H705P ++ 1803/1804 G181N/A548P/H705P +++ 1805/1806A631/E536L + 1807/1808 A548P/H705P + 1809/1810 L580M + 1811/1812E536L/A548P/H699F + 1813/1814 G181N/E536L/A548P/H705M + 1815/1816G181N/E536L/A548P ++ 1817/1818 E536L/A548P + 1819/1820 E536L ++^(a)Levels of increased production were determined relative to thereference polypeptide of SEQ ID NO: 1764, and are defined as follows:“+” = production at least 1.5-fold, but less than 2.4-fold; “++” = atleast 2.4-fold, but less than 3.0-fold; and “+++” = at least 3.0-foldincreased production, relative to the reference polypeptide.Shake Flask Powder Characterization Assay and Analysis for GlucosylTransfer from Sucrose to Rebaudioside A

An experiment was performed to characterize the activity of theengineered round 9 SUS variants on sucrose and ADP to facilitate theformation of rebaudioside D from rebaudioside A. Shake flask powder(SFP) was added to a 100 μL total reaction volume at 0.006-0.2 μLconcentration containing 50 mM potassium phosphate buffer, pH6, 15 mMrebaudioside A (>97% purity), 45 mM sucrose, 9 mM fructose, 0.2 mM ADP,and 1 g/L GT SEQ ID NO: 3244. The reaction was performed at 50° C. in aThermotron® titre-plate shaker with 300 RPM shaking for 2 h withoutpre-incubation, or the SFP was pre-incubated at 10× final concentrationin pH 6 potassium phosphate buffer at 62° C. for 1 h prior to 60° C.reaction for 2h. The reaction was solubilized by diluting 40× intowater, quenched by diluting 10× into acetonitrile with 0.2% formic acid,and precipitated by centrifugation. The supernatant was diluted 3.3× inwater and analyzed for steviol glycosides by RapidFire SPE-MS/MS, withthe instrument and parameters described in Table 31.1. All 8 of thevariants listed in Table 35.2 had higher activities than SEQ ID NO:1764. The variant with the mutations G181N, A548P, H705P (SEQ ID NO:1804), relative to SEQ ID NO: 1764, had the most beneficial mutationsand was selected for further directed evolution for the catalysis of therecycling reaction transferring aglucosyl from sucrose to ADP.

TABLE 35.2 SUS Round 9 SFP Variants and RebD Levels Increased IncreasedSEQ ID NO: Amino Acid Differences RebD, RebD, (nt/aa) (Relative to SEQID NO: 1764) 50° C.^(a) 60° C.^(a) 1787/1788 V681A ++ +++ 1791/1792V270L/V681A ++ +++ 1793/1794 G117E/A122D/V270L/L540M/ + + V681A1795/1796 L407T/V681A + +++ 1803/1804 G181N/A548P/H705P ++ +++ 1807/1808A548P/H705P ++ +++ 1809/1810 L580M + + 1815/1816 G181N/E536L/A548P + +++^(a)Levels of increased production were determined relative to thereference polypeptide of SEQ ID NO: 1764, and are defined as follows:“+” = production at least 1.1-fold, but less than 1.7-fold; “++” =production at least 1.7-fold, but less than 2.3-fold; and “+++” =production at least 2.3-fold greater, as compared to the referencepolypeptide.

Example 36 Sucrose Synthase Variants of SEQ ID NO: 1804

Directed evolution of the sucrose synthase encoded by SEQ ID NO: 1803was continued by constructing libraries of variant genes in whichmutations associated with improved activity in earlier rounds ofevolution were recombined and in which certain structural features weresubjected to saturation mutagenesis. These libraries were then plated,grown, and screened using the high-throughput (HTP) assay describedbelow, to provide a tenth round (“Round 10”) of 82 engineered SuSvariant polypeptides with increased activity toward the generation ofADP-glucose.

HTP Coupled Assay for Glucose Transfer from Sucrose to ADP toRebaudioside A

Libraries were screened using the following HTP enzyme-coupled assay.Pelleted E. coli cultures were lysed with 400 μL of Tris-HCl, pH 7.5,with 1 mM magnesium sulfate and 0.5 mg/mL lysozyme and polymyxin Bsulfate (PMBS) and cleared by centrifugation. Lysate was diluted 20×into potassium phosphate buffer, pH 6.0, and pre-incubated for 15minutes at 64° C. (combinatorial libraries) or 65° C. (saturationmutagenesis library). Then, 10 μL diluted SuS lysate and 0.5-1 g/L GTSEQ ID NO: 3696 or 3502, respectively, were used in 100 μL reactionvolume with 15 mM rebaudioside A (>97% purity), 0.2 mM ADP(Sigma, >93%), 45 mM sucrose (cane sugar), and 9 mM fructose. Thefollowing reaction conditions were used: 50 mM potassium phosphatebuffer, pH 6, 60° C. in a Thermotron® titre-plate shaker with 300 RPMshaking for 3 h. The reaction described above was solubilized bydiluting 40× in water, quenched by diluting 10× in acetonitrile with0.2% formic acid, and precipitated by centrifugation. The supernatantwas diluted 3.3× in water and steviol glycoside products were detectedby RapidFire SPE-MS/MS, with the instrument and parameters described inTable 31.1. After analysis, the engineered SuS variant polypeptides thatshowed improved activity coupled with a GT on rebaudioside A wereidentified and are listed in Table 36.1. Shake-flask scale cultures weregrown for protein characterization as described in Example 31 forvariants with the amino acid mutations shown in Table 36.2.

TABLE 36.1 SUS Round 10 Variants and RebD Levels SEQ ID NO: Amino AcidDifferences Increased (nt/aa) (Relative to SEQ ID NO: 1804) RebD^(a)1821/1822 N347R/Q550I/V681A + 1823/1824N347R/S532Y/Q550I/T640N/V681A/H699F + 1825/1826 N347R/V681A + 1827/1828N347R/Y434H/P517A/L562I/T640N/V681A + 1829/1830N347R/P517A/S532Y/V681A + 1831/1832 N347R/Y434H/V681A + 1833/1834E536L/L580M/V681A + 1835/1836 N347R/Q550I/L580M/V681A + 1837/1838N347R/E536L/L562I/V681A + 1839/1840 P517A/V681A + 1841/1842S532Y/V681A + 1843/1844 Y434H/P517A/S532Y/V681A + 1845/1846N347R/Y434H/S532Y/L562I/T640N/V681A + 1847/1848 L562I/V681A + 1849/1850N347R/Y434H/Q550I/L562I/V681A + 1851/1852 A389G ++ 1853/1854 S539A +++1855/1856 L433K ++ 1857/1858 R71Q +++ 1859/1860 E87L ++ 1861/1862 P13K++ 1863/1864 D765A ++ 1865/1866 Q37R ++ 1867/1868 R90H ++ 1869/1870D60L + 1871/1872 S531T ++ 1873/1874 Q37G + 1875/1876 S539R +++ 1877/1878D17R +++ 1879/1880 P535S + 1881/1882 R606H ++ 1883/1884 V98L + 1885/1886L388K ++ 1887/1888 P57W +++ 1889/1890 P535H + 1891/1892 P13H + 1893/1894E727K + 1895/1896 V85H + 1897/1898 R606A ++ 1899/1900 A789R + 1901/1902A789N + 1903/1904 A18G + 1905/1906 P13N + 1907/1908 K415H + 1909/1910S531R +++ 1911/1912 R711K +++ 1913/1914 R606Q ++ 1915/1916 R606L +1917/1918 R606M ++ 1919/1920 R6061 + 1921/1922 D52P +++ 1923/1924 D52W +1925/1926 S30H + 1927/1928 E87H ++ 1929/1930 A118N ++ 1931/1932 Q769K+++ 1933/1934 E129G +++ 1935/1936 D52R ++ 1937/1938 E99I + 1939/1940E129A + 1941/1942 L433P ++ 1943/1944 E748T + 1945/1946 A164T + 1947/1948A707G ++ 1949/1950 P57R ++ 1951/1952 A608P ++ 1953/1954 A118G +1955/1956 P535A + 1957/1958 D180P + 1959/1960 Q769R + 1961/1962 N183P+++ 1963/1964 R71G ++ 1965/1966 R606V ++ 1967/1968 E129T +++ 1969/1970A164S ++ 1971/1972 L388R ++ 1973/1974 H365W + 1975/1976 D52G +++1977/1978 G589S +++ 1979/1980 E738S +++ 1981/1982 D765S +++ 1983/1984S531A ++ ^(a)Levels of increased production were determined relative tothe reference polypeptide of SEQ ID NO: 1804, and are defined asfollows: “+” = production at least 1.3-fold, but less than 2-fold; “++”= at least 2-fold, but less than 2.36-fold; and “+++” = at least2.36-fold increased production, relative to the reference polypeptide.Shake Flask Powder Characterization Assay and Analysis for GlucosylTransfer from Sucrose to Rebaudioside A

An experiment was performed to characterize the activity of theengineered round 10 SUS variants on sucrose and ADP to facilitate theformation of rebaudioside D from rebaudioside A. Shake flask powder(SFP) was made up to 0.06-2 g/L in potassium phosphate buffer, pH6, andan aliquot was pre-incubated at 64° C. in a thermocycler for 15 minutes.Then, 10 μLof these SFP dilutions, either pre-incubated or notpre-incubated, were added to a 100 μL total reaction volume containing50 mM potassium phosphate buffer, pH 6, 15 mM rebaudioside A (>97%purity), 45 mM sucrose, 9 mM fructose, 0.2 m ADP, and g/L GT SEQ IDNO:3696. The reactions were performed at 55° C. for SFP samples thatwere not pre-incubated and at 60° C. for pre-incubated SFP in aThermotron® titre-plate shaker with 300 RPM shaking for 3h. The reactionwas solubilized by diluting 40× into water, quenched by diluting 10×into acetonitrile with 0.2% formic acid, and precipitated bycentrifugation. The supernatant was diluted 3.3× in water and analyzedfor steviol glycosides by RapidFire SPE-MS/MS, with the instrument andparameters described in Table 31.1. All 7 of the variants listed inTable 36.2 had higher activities than SEQ ID NO: 1804 under thepre-incubated condition, and all but one also had higher activity underthe 55° C. condition. The variant with the mutations P517A and V681A(SEQ ID NO: 1840) was the most improved under both conditions relativeto SEQ ID NO: 1804, so it was selected for further directed evolutionfor the catalysis of the recycling reaction transferring a glucose fromsucrose to ADP.

TABLE 36.2 SUS Round 10 SFP Variants and RebD Levels SEQ ID IncreasedIncreased NO: Amino Acid Differences (Relative RebD RebD (nt/aa) to SEQID NO: 1804) 55° C.^(a) 60° C.^(a) 1821/1822 N347R/Q550I/V681A + ++1823/1824 N347R/S532Y/Q550I/T640N/ − + V681A/H699F 1827/1828N347R/Y434H/P517A/L562I/ + ++ T640N/V681A 1831/1832 N347R/Y434H/V681A +++ 1837/1838 N347R/E536L/L562I/V681A + ++ 1839/1840 P517A/V681A ++ +++1845/1846 N347R/Y434H/S532Y/L562I/ + ++ T640N/V681A ^(a)Levels ofincreased production were determined relative to the referencepolypeptide of SEQ ID NO: 1804, and are defined as follows: “−” =production less than that of the reference polypeptide; “+” = productionat least that of the reference polypeptide, but less than 1.5-fold; “++”= production at least 1.5-fold, but less than 2-fold; and “+++” =production at least 2-fold greater, as compared to the referencepolypeptide.

Example 37 Sucrose Synthase Variants of SEQ ID NO: 1840

Directed evolution of the sucrose synthase encoded by SEQ ID NO: 1839was continued by constructing libraries of variant genes in whichmutations associated with improved activity in earlier rounds ofevolution were recombined and in which certain structural features weresubjected to saturation mutagenesis. These libraries were then plated,grown, and screened using the high-throughput (HTP) assay describedbelow, to provide an eleventh round (“Round 11”) of 167 engineered SuSvariant polypeptides with increased activity toward the generation ofADP-glucose.

HTP Coupled Assay for Glucose Transfer from Sucrose to ADP toRebaudioside A

Libraries were screened using the following HTP enzyme-coupled assay.Pelleted E. coli cultures were lysed with 400 μL of Tris-HCl, pH 7.5,with 1 mM magnesium sulfate and 0.5 mg/mL lysozyme and polymyxin Bsulfate (PMBS), and cleared by centrifugation. Lysate was diluted 10-20×into potassium phosphate buffer, pH 6.0, and pre-incubated for 15minutes at 66° C. Then, 10 μL diluted, pre-incubated SuS lysate and 0.5g/L GT SEQ ID NO: 3696 or 3956, were used in 100 μL reaction volume with15 mM rebaudioside A (>97% purity), 0.2 mM ADP (Amresco, >93%), 45 mMsucrose (cane sugar), and 9 mM fructose. The following reactionconditions were used: 50 mM potassium phosphate buffer, pH 6, 60° C., ina Thermotron® titre-plate shaker with 300 RPM shaking for 4 h. Thereaction described above was solubilized by diluting 40× in water,quenched by diluting 5-10× in acetonitrile with 0.2% formic acid, andprecipitated by centrifugation. The supernatant was diluted in water to˜10 μM steviol glycosides and steviol glycoside products were detectedby RapidFire SPE-MS/MS with the instrument and parameters described inTable 31.1. After analysis, the engineered SuS variant polypeptides thatshowed improved activity coupled with a GT on rebaudioside A wereidentified and are listed in Tables 37.1 and 37.2. Shake-flask scalecultures were grown for protein characterization as described in Example31 for variants with the amino acid mutations shown in Table 37.3.

TABLE 37.1 SUS Round 11 Combinatorial Variants and RebD Levels SEQ IDNO: Amino Acid Differences Increased (nt/aa) (Relative to SEQ ID NO:1840) RebD^(a) 1985/1986 D17R/E129T/G589S + 1987/1988 D17R/G589S/E738S++ 1989/1990 D17R/E129T/G589S/E738S + 1991/1992D17R/E129T/G589S/E738S/D765S +++ 1993/1994 D52P/E129T/G589S/E738S/D765S+++ 1995/1996 D52P/E129T/E738S + 1997/1998 D52P/A118N/E129T/D765S +1999/2000 D17R/D52P/E87H/G589S/E738S/D765S +++ 2001/2002E87H/E129T/L388K/G589S + 2003/2004 D17R/D52G/E129T/R653H/E738S/D765S +2005/2006 D17R/E738S/D765S + 2007/2008D17R/D52G/E87H/E129T/L388K/G589S/E738S ++ 2009/2010D17R/L388K/G589S/E738S ++ 2011/2012 D17R/D52P/E129T/E738S ++ 2013/2014D17R/D52P/E129T/G589S/D765S ++ 2015/2016D17R/D52P/E87H/E129G/L388K/G589S +++ 2017/2018D52G/A118N/L388K/G589S/E738S +++ 2019/2020D52P/E87H/A118N/E129T/L388K/D765S ++ 2021/2022D52G/G84A/E129T/L388K/E738S/D765S + 2023/2024D17R/D52P/A118N/E129T/S265T/G589S/D765S ++ 2025/2026D17R/D52P/A118N/L388K/G589S/E738S/D765S +++ 2027/2028D17R/D52G/E129G/G589S/E738S + 2029/2030D17R/D52G/E129T/L388K/G589S/E738S +++ 2031/2032D17R/E87H/A118N/L388K/E738S + 2033/2034 D52G/L388K/E738S/D765S +2035/2036 D17R/D52G/A118N/L388K/E738S/D765S + 2037/2038D17R/D52P/E129G/E738S/D765S + 2039/2040D17R/D52P/E87H/A118N/E129T/G589S/E738S/D765S + 2041/2042D52P/E87H/A118N/L388K/G589S/E738S ++ 2043/2044G84A/E129T/G589S/E738S/D765S + 2045/2046 D17R/D52G/G589S/D765S +2047/2048 D17R/D52P/G589S/E738S/D765S +++ 2049/2050D17R/D52G/E87H/E129T/E738S + 2051/2052D17R/D52G/E87H/A118N/E129T/G589S/D765S ++ 2053/2054D17R/D52P/A118N/E129T/G589S/E738S/D765S ++ 2055/2056 D52G/E87H/E738S +2057/2058 D17R/D52G/E87H/A118N/E129G/G589S/E738S + 2059/2060D17R/D52P/E129G/L388K/G589S/E738S/D765S ++ 2061/2062D17R/D52P/E87H/A118N/E129T/L388K/G589S/E738S/D765S + 2063/2064D17R/D52P/L388K/G589S/E738S/D765S ++ 2065/2066D17R/D52P/E87H/E129T/L388K/G589S/E738S + 2067/2068D52G/E87H/E129T/L388K/E738S/D765S + 2069/2070D17R/D52G/E87H/L388K/G589S/D765S + 2071/2072D17R/D52P/A118N/L388K/G589S/E738S ++ 2073/2074 D17R/G589S/D765S +2075/2076 D52G/E87H/A118N/G589S/E738S/D765S ++ 2077/2078D52G/E738S/D765S + 2079/2080 D52P/G589S/E738S/D765S ++ 2081/2082D52P/E87H/E129G/D765S +++ 2083/2084 D17R/E129T/E738S/D765S ++ 2085/2086D17R/D52P/L388K/G589S/E738S +++ 2087/2088D17R/A118N/E129T/L388K/E738S/D765S + 2089/2090D52P/A118N/E129T/G589S/E738S ++ 2091/2092 D52G/A118N/L388K/E738S/D765S +2093/2094 G589S/E738S/D765S ++ 2095/2096D17R/D52G/A118N/E129T/L388K/G589S/E738S/D765S ++ 2097/2098D52G/E129G/G589S/D765S + 2099/2100 D52P/G589S/E738S +++ 2101/2102D52G/G589S/E738S/D765S +++ 2103/2104 D52P/E87H/G589S/E738S +++ 2105/2106D17R/D52P/A118N/E129T/E738S/D765S + 2107/2108 D17R/D52G/E129T/G589S +2109/2110 D17R/E87H/E129T/L388K/E738S + 2111/2112E87H/A118N/E129T/D765S + ^(a)Levels of increased production weredetermined relative to the reference polypeptide of SEQ ID NO: 1840, anddefined as follows: “+” = production at least 11-fold, but less than14.9-fold; “++” = at least 14.9-fold, but less than 18.3-fold; and “+++”= at least 18.3-fold increased production, relative to the referencepolypeptide.

TABLE 37.2 SUS Round 11 Saturation Mutagenesis Variants and RebD LevelsSEQ ID NO: Amino Acid Differences Increased (nt/aa) (Relative to SEQ IDNO: 1840) RebD^(a) 2113/2114 R14K ++ 2115/2116 I480V ++ 2117/2118 M263Y++ 2119/2120 D218V + 2121/2122 H154A +++ 2123/2124 G360R + 2125/2126E534W/E739K +++ 2127/2128 G603H + 2129/2130 E59N/D72N ++ 2131/2132Q33S + 2133/2134 V134P ++ 2135/2136 P81I +++ 2137/2138 S393H ++2139/2140 V134A ++ 2141/2142 E390M + 2143/2144 P364S + 2145/2146 Y434R +2147/2148 W212Y + 2149/2150 S15I + 2151/2152 V316T + 2153/2154 P530F +++2155/2156 H92G + 2157/2158 S175G +++ 2159/2160 G652L +++ 2161/2162 E534R+++ 2163/2164 G603S +++ 2165/2166 S26I + 2167/2168 H154R + 2169/2170E105S + 2171/2172 E59R + 2173/2174 D130Y +++ 2175/2176 F542W ++2177/2178 S26E + 2179/2180 S15P + 2181/2182 W79Y ++ 2183/2184 P81L +++2185/2186 A165L + 2187/2188 P81G ++ 2189/2190 I362E + 2191/2192 G603A ++2193/2194 E534W +++ 2195/2196 G652S ++ 2197/2198 D218T ++ 2199/2200D218A ++ 2201/2202 L20M + 2203/2204 R319S + 2205/2206 S15A + 2207/2208E59W + 2209/2210 E59S + 2211/2212 G652T +++ 2213/2214 L241T ++ 2215/2216D218N ++ 2217/2218 H154E + 2219/2220 Y434G ++ 2221/2222 Q46V + 2223/2224Q46R + 2225/2226 W79H ++ 2227/2228 G652K +++ 2229/2230 Q33H/H154C +++2231/2232 M263S + 2233/2234 A165I + 2235/2236 V93T + 2237/2238 G603E +++2239/2240 Q349R ++ 2241/2242 I480P ++ 2243/2244 E534L +++ 2245/2246E534G +++ 2247/2248 E59C + 2249/2250 A213V + 2251/2252 Q33P + 2253/2254Q46T + 2255/2256 E534K +++ 2257/2258 E59A ++ 2259/2260 Q46I + 2261/2262L58M + 2263/2264 E534T +++ 2265/2266 G50R + 2267/2268 G652R +++2269/2270 S256G ++ 2271/2272 A97V/H154S ++ 2273/2274 V316H ++ 2275/2276V24S + 2277/2278 G360E + 2279/2280 E498Q + 2281/2282 G360D + 2283/2284Q349T ++ 2285/2286 S175T + 2287/2288 G603Q ++ 2289/2290 A18V/I362A ++2291/2292 H154S + 2293/2294 D104T + 2295/2296 S26A ++ 2297/2298 Q46G +2299/2300 Q33L + 2301/2302 D218S ++ 2303/2304 A165T + 2305/2306 Q349D +2307/2308 D54M + 2309/2310 S26T + 2311/2312 E498L + 2313/2314 M185L +2315/2316 D218Q ++ 2317/2318 E59V ++ ^(a)Levels of increased productionwere determined relative to the reference polypeptide of SEQ ID NO:1840, and defined as follows: “+” = production at least 1.2-fold, butless than 1.62-fold; “++” = at least 1.62-fold, but less than 2.4-fold;and “+++” = at least 2.4-fold increased production, relative to thereference polypeptide.Shake Flask Powder Characterization Assay and Analysis for GlucosylTransfer from Sucrose to Rebaudioside A

An experiment was performed to characterize the activity of theengineered round 11 SUS variants on sucrose and ADP to facilitate theformation of rebaudioside D from rebaudioside A. Shake flask powder(SFP) was made up to 0.03-1 g/L in potassium phosphate buffer, pH 6, andan aliquot was pre-incubated at 66° C. in a thermocycler for 15 minutes.Then, 10 μL of these SFP dilutions, either pre-incubated or notpre-incubated, were added to a 100 μL total reaction volume containing50 mM potassium phosphate buffer, pH 6, 15 mM rebaudioside A (>97%purity), 45 mM sucrose, 9 mM fructose, 0.2 mM ADP, and 0.5 g/L GT SEQ IDNO: 3956. The reactions were performed at 55° C. for SFP samples thatwere not pre-incubated and at 60° C. for pre-incubated SFP in aThermotron® titre-plate shaker with 300 RPM shaking for 4 h. Thereaction was solubilized by diluting 40× into water, quenched bydiluting 10× into acetonitrile with 0.2% formic acid, and precipitatedby centrifugation. The supernatant was diluted 3.3× in water andanalyzed for steviol glycosides by RapidFire SPE-MS/MS with theinstrument and parameters described in Table 31.1. All 8 of the variantslisted in Table 37.3 had higher activities than SEQ ID NO: 1840 underthe pre-incubated condition, and all but three also had higher activityunder the 55° C. condition. The variant with the mutations D17R, D52P,L388K, G589S, E738S, and D765S (SEQ ID NO: 2064), which was improvedunder both conditions relative to SEQ ID NO: 1840, was selected forfurther directed evolution for the catalysis of the recycling reactiontransferring a glucose from sucrose to ADP.

TABLE 37.3 SUS Round 11 SFP Variants and RebD Levels SEQ ID IncreasedIncreased NO: Amino Acid Differences RebD, RebD, (nt/aa) (Relative toSEQ ID NO: 1840) 55° C.^(a) 60° C.^(a) 1999/2000D17R/D52P/E87H/G589S/E738S/ − ++ D765S 2017/2018D52G/A118N/L388K/G589S/E738S − ++ 2025/2026 D17R/D52P/A118N/L388K/G589S/− ++ E738S/D765S 2047/2048 D17R/D52P/G589S/E738S/D765S + +++ 2059/2060D17R/D52P/E129G/L388K/G589S/ + +++ E738S/D765S 2063/2064D17R/D52P/L388K/G589S/E738S/ + +++ D765S 2099/2100 D52P/G589S/E738S + ++2103/2104 D52P/E87H/G589S/E738S + ++ ^(a)Levels of increased productionwere determined relative to the reference polypeptide of SEQ ID NO:1840, and defined as follows: “−” = production less than that of thereference polypeptide; “+” = production at least that of the referencepolypeptide, but less than 2-fold; “++” = production at least 2-fold,but less than 3.2-fold; and “+++” = production at least 3.2-fold greaterthan that of the reference polypeptide.

Example 38 Sucrose Synthase Variants of SEQ ID NO: 2064

Directed evolution of the sucrose synthase encoded by SEQ ID NO: 2063,was continued by constructing libraries of variant genes in whichmutations associated with improved activity in earlier rounds ofevolution were recombined and in which certain structural features weresubjected to saturation mutagenesis. These libraries were then plated,grown, and screened using the high-throughput (HTP) assay describedbelow, to provide a twelfth round (“Round 12”) of 92 engineered SuSvariant polypeptides with increased activity toward the generation ofADP-glucose.

HTP Coupled Assay for Glucose Transfer from Sucrose to ADP toRebaudioside A

Libraries were screened using the following HTP enzyme-coupled assay.Pelleted E. coli cultures were lysed with 400 μL of Tris-HC, pH 7.5,with 1 mM magnesium sulfate and 0.5 mg/mL lysozyme and polymyxin Bsulfate (PMBS), and cleared by centrifugation. Lysate was diluted 10×into potassium phosphate buffer, pH 6.0, and pre-incubated for 15minutes at 68° C. Then, 10 μL diluted, pre-incubated SuS lysate and 0.5g/L GT SEQ ID NO: 3956 or 4256 were used in 100 μL reaction volume with15 mM rebaudioside A (>97% purity), 0.2 mM ADP (Amresco, >93%), 37.5 mMsucrose (cane sugar), and 9 mM fructose. The following reactionconditions were used: 50 mM potassium phosphate buffer, pH 6, 60° C. ina Thermotron® titre-plate shaker with 300 RPM shaking for 4h. Thereaction described above was solubilized by diluting 40× in water,quenched by diluting 5× in acetonitrile with 0.2% formic acid, andprecipitated by centrifugation. The supernatant was diluted 7.5× inwater and steviol glycoside products were detected by RapidFireSPE-MS/MS with the instrument and parameters described in Table 31.1.After analysis, the engineered SuS variant polypeptides that showedimproved activity coupled with a GT on rebaudioside A were identifiedand are listed in Tables 38.1 and 38.2. Shake-flask scale cultures weregrown for protein characterization as described in Example 31 forvariants with the amino acid mutations shown in Table 38.3.

TABLE 38.1 SUS Round 12 Combinatorial Variants and RebD Levels SEQ IDNO: Amino Acid Differences Increased (nt/aa) (Relative to SEQ ID NO:2064) RebD^(a) 2319/2320 P57W/S531R/S532Y/E536L/L562I ++ 2321/2322E129T/Q550I/L562I + 2323/2324R71Q/E129T/D180P/Y434H/S532Y/E536L/S539R/R711K/A789N + 2325/2326E87H/N347R/E536L/S539R/Q550I + 2327/2328P57W/N347R/Y434H/S531R/S532Y/S539R/A789N + 2329/2330P57W/E129G/E536L/R606M/A789N + 2331/2332 R71Q/E129T/R606M + 2333/2334N347R/S531R/Q550I/R711K ++ 2335/2336 P57W/E87H/N347R/L562I + 2337/2338N347R/E536L/L562I/R606M/E612A + 2339/2340 E129T/E536L/S539R/L562I +2341/2342 S531R/S532Y/E536L/Q550I/L562I/R606M/A789N + 2343/2344E129T/D180P/Y434H/L562I/R711K/A789N + 2345/2346 P57W/R90H/E129G/L562I +2347/2348 P57W/R71Q/E129G/S531R/S532Y/E536L/S539R/R606H ++ 2349/2350R90H/E129T/S539R/Q550I/R606H + 2351/2352 R71Q/E129T/S531R + 2353/2354P57W/L562I/R606M/R711K ++ 2355/2356Y434H/S531R/S539R/Q550I/L562I/R711K + 2357/2358P57W/E129G/S531R/S539R/L562I/A789N + 2359/2360P57W/E129T/N347R/E536L/Q550I/L5621/R711K/A789N + 2361/2362P57W/E129T/R606M + 2363/2364 E129T/D180P/R606M/R711K/A789N + 2365/2366P57W/R90H/E129T/L562I/R711K ++ 2367/2368 E129G/Q550I + 2369/2370P57W/R71Q/L562I/R606M/R711K/A789N + 2371/2372E87H/G189D/S532Y/E536L/L5621/R711K/A789N + 2373/2374 L5621/R711K ++2375/2376 P57W/R71Q/E129T/D180P/Y434H/E536L/L562I ++ 2377/2378P57W/R71Q/E87H/N347R/Y434H/L562I/R606H +++ 2379/2380R71Q/E536L/S539R/L562I +++ 2381/2382 P57W/E129T/N347R/Q550I/R711K ++2383/2384 P57W/L96Q/E129T/D180P/S531R/S532Y/Q550I/L562I ++ 2385/2386E87H/N347R/S531R/R606M/A789N + 2387/2388 D180P/S532Y + 2389/2390S531R/S532Y/E536L/S539R/L562I/R711K/A789N +++ 2391/2392R71Q/N347R/S532Y/Q550I/L562I/R711K +++ 2393/2394P57W/D180P/L5621/R606H/E612A + 2395/2396 P57W/R71Q/A789N + 2397/2398P57W/Y434H/Q550I/L562I/R606H/E612A/A789N ++ 2399/2400P57W/E87H/D180P/S531R/S532Y/L562I/R606M/E612A/R711K ++ 2401/2402S531R/S532Y/L562I/R606H/R711K ++ 2403/2404 E129G/L562I/R606M/R711K +++2405/2406 P57W/R71Q/E129T/Y434H/S531R/E536L/L562I +++ 2407/2408N347R/Q550I/L562I/R606H +++ 2409/2410 E129T/N347R/L562I ++ 2411/2412P57W/D180P/L562I ++ 2413/2414 E129T/S539R/L562I/A789N ++ 2415/2416N347R/E536L/S539R/Q550I/R711K/A789N ++ 2417/2418 D180P/Q550I/R606M +++2419/2420 Y434H/Q550I +++ 2421/2422P57W/E129T/N347R/S531R/S532Y/S539R/L5621/R711K/A747V + 2423/2424R71Q/N347R/E536L/L562I/E612A/A789N ++ 2425/2426R71Q/E129T/D180P/N347R/S531R/S539R/Q550I + 2427/2428 S532Y/S539R/Q550I+++ 2429/2430 E129G/Q550I/L562I +++ 2431/2432 P57W/L562I/R711K +^(a)Levels of increased production were determined relative to thereference polypeptide of SEQ ID NO: 2064, and defined as follows: “+” =production at least 15-fold, but less than 38.25-fold; “++” = at least38.25-fold, but less than 50.3-fold increased production; and “+++” = atleast 50.3-fold increased production, relative to the referencepolypeptide.

TABLE 38.2 SUS Round 12 Saturation Mutagenesis Variants and RebD LevelsSEQ ID NO: Amino Acid Differences (nt/aa) (Relative to SEQ ID NO: 2064)Increased RebD^(a) 2433/2434 V641L + 2435/2436 L688H + 2437/2438 Q487K+++ 2439/2440 A41K ++ 2441/2442 F684M ++ 2443/2444 L186V + 2445/2446I91G + 2447/2448 G112Q ++ 2449/2450 V89M + 2451/2452 Q487T ++ 2453/2454F684G + 2455/2456 F684T + 2457/2458 A25T/G112W ++ 2459/2460 P226V +2461/2462 L764R +++ 2463/2464 L688G ++ 2465/2466 L688A +++ 2467/2468V763L + 2469/2470 F684H ++ 2471/2472 S330A + 2473/2474 R21Q + 2475/2476G112R + 2477/2478 D259G + 2479/2480 V200A + 2481/2482 Q487V + 2483/2484Q487R ++ 2485/2486 L688Q +++ 2487/2488 P318A + 2489/2490 G485A ++2491/2492 G485S ++ 2493/2494 V89L + 2495/2496 I91C ++ 2497/2498 L688F+++ 2499/2500 Q487I +++ 2501/2502 S674A + ^(a)Levels of increasedproduction were determined relative to the reference polypeptide of SEQID NO: 2064, and defined as follows: “+” = production at least 2-fold,but less than 3.7-fold; “++” = at least 3.7-fold, but less than6.4-fold; and “+++” = at least 6.4-fold increased production, relativeto the reference polypeptide.Shake Flask Powder Characterization Assay and Analysis for GlucosylTransfer from Sucrose to Rebaudioside A

An experiment was performed to characterize the activity of theengineered round 12 SUS variants on sucrose and ADP to facilitate theformation of rebaudioside D from rebaudioside A. Shake flask powder(SFP) was made up to 0.03-1 g/L in potassium phosphate buffer, pH6, andan aliquot was pre-incubated at 68° C. in a thermocycler for 15 minutes.Then, 10 μLof these SFP dilutions, either pre-incubated or notpre-incubated, were added to a 100 μL total reaction volume containing50 mM potassium phosphate buffer, pH6, 15 mM rebaudioside A (>97%purity), 37.5 mM sucrose, 9 mM fructose, 0.2 mM ADP, and 0.5 g/L GT SEQID NO: 4256. The reactions were performed at 55° C. for SFP samples thatwere not pre-incubated and at 60° C. for pre-incubated SFP in aThermotron® titre-plate shaker with 300 RPM shaking for 4h. The reactionwas solubilized by diluting 40× into water, quenched by diluting 10×into acetonitrile with 0.2% formic acid, and precipitated bycentrifugation. The supernatant was diluted 3.3× in water and analyzedfor steviol glycosides by RapidFire SPE-MS/MS with the instrument andparameters described in Table 31.1. A further thermostabilitycharacterization was conducted with the clarified shake flask lysatesprior to lyophilization as follows: lysates were diluted 400× in bufferand incubated in a thermocycler at a gradient of 55-70° C. for 16-18 h.To determine the % activity remaining, the pre-incubated lysates wereassayed as described above with either stevioside or rebaudioside D and4h incubation at 55° C. The percent activity remaining was expressed asactivity at high temperature divided by activity at lowest pre-incubatedtemperature. All 7 of the variants listed in Table 38.3 had higheractivities than SEQ ID NO: 2064 under the pre-incubated conditions, andtwo also were less deleterious under the 55° C. condition. The variantwith the mutations P57W, L562, and R711K (SEQ ID NO:2432), which wasimproved under all conditions relative to SEQ ID NO: 2064 and containedthe top mutation from the round 10 saturation mutagenesis screen, wasselected for further directed evolution for the catalysis of therecycling reaction transferring a glucose from sucrose to ADP.

TABLE 38.3 SUS Round 12 SFP Variants and RebD Levels SEQ ID Amino AcidDifferences Increased Increased % Activity NO: (Relative to SEQ ID RebD,RebD, Re- (nt/aa) NO: 2064) 55° C.^(a) 60° C.^(a) maining^(b) 2333/2334N347R/S531R/Q550I/ − ++ ++ R711K 2373/2374 L562I/R711K − +++ + 2389/2390S531R/S532Y/E536L/ − ++ ++ S539R/L562I/R711K/ A789N 2405/2406P57W/R71Q/E129T/ − ++ +++ Y434H/S531R/E536L/ L562I 2427/2428S532Y/S539R/Q550I + +++ ++ 2429/2430 E129G/Q550I/L562I − ++ ++ 2431/2432P57W/L562I/R711K + +++ ++ ^(a)Levels of increased production weredetermined relative to the reference polypeptide of SEQ ID NO: 2064, anddefined as follows: “−” = production less than 0.8-fold that of thereference polypeptide; “+” = production at least 0.8-fold, but less than1.2-fold; “++” = production at least 1.2-fold, but less than 4.7-fold;and “+++” = production at least 4.7-fold increased, relative to that ofthe reference polypeptide. ^(b)The percent of activity remaining foreach variant was determined following 16-18 h pre-incubation at 65° C.,relative to the production of each variant following pre-incubation at55° C. and is defined as follows: “−” = less than 55% of activityremained following pre-incubation at 55° C.; “+” = at least 55%activity, but less than 65% activity remained; “++” = at least 65%activity, but less than 75% activity remained; and “+++” = at least 75%activity remained.

Example 39 Sucrose Synthase Variants of SEQ ID NO: 2432

Directed evolution of the sucrose synthase encoded by SEQ ID NO: 2431,was continued by constructing libraries of variant genes in whichmutations associated with improved activity in earlier rounds ofevolution were recombined and in which certain structural features weresubjected to saturation mutagenesis. These libraries were then plated,grown, and screened using the high-throughput (HTP) assay describedbelow, to provide a thirteenth round (“Round 13”) of 46 engineered SuSvariant polypeptides with increased activity toward the generation ofADP-glucose.

HTP Coupled Assay for Glucose Transfer from Sucrose to ADP toRebaudioside A

Libraries were screened using the following HTP enzyme-coupled assay.Pelleted E. coli cultures were lysed with 400 μL of Tris-HCl, pH 7.5,with 1 mM magnesium sulfate and 0.5 mg/mL lysozyme and polymyxin Bsulfate (PMBS) and cleared by centrifugation. For the combinatoriallibrary, lysate was diluted 10× into potassium phosphate buffer, pH 6.0,and pre-incubated for 15 minutes at 73° C. For the saturationmutagenesis library, lysate was diluted 20× into potassium phosphatebuffer, pH 6.0, and pre-incubated for 17.5 hours at 62° C. Then, 10 μLdiluted, pre-incubated SuS lysate and 0.5 g/L GT SEQ ID NO: 4256 or 4550were used in 100 μL reaction volume with 15 mM rebaudioside A (>97%purity), 0.2 mM ADP (Amresco, >93%), 37.5 mM sucrose (cane sugar), and 9mM fructose. The following reaction conditions were used: 50 mMpotassium phosphate buffer, pH 6, 60° C. in a Thermotron® titre-plateshaker with 300 RPM shaking for 3-4 h. The reaction described above wassolubilized by diluting 40× in water, quenched by diluting 5× inacetonitrile with 0.2% formic acid, and precipitated by centrifugation.The supernatant was diluted 7.5× in water and steviol glycoside productswere detected by RapidFire SPE-MS/MS with the instrument and parametersdescribed in Table 31.1. After analysis, the engineered SuS variantpolypeptides that showed improved activity coupled with a GT onrebaudioside A were identified and are listed in Tables 39.1 and 39.2.Shake-flask scale cultures were grown for protein characterization asdescribed in Example 31 for variants with the amino acid mutations shownin Table 39.3.

TABLE 39.1 SUS Round 13 Combinatorial Variants and RebD Levels SEQ IDNO: Amino Acid Differences Increased (nt/aa) (Relative to SEQ ID NO:2432) RebD^(a) 2503/2504 Q33H/H154A/I480P/P530F/E534W/G603Q/R606M +2505/2506 L58M/E59A/W79H/S175G/1480P/E534W/Q550I/G652R + 2507/2508Q33H/L58M/E59A/P81G/D130Y/1480V/P530F/E534W/Q550I/ ++ G652K 2509/2510Q33H/L47P/E59A/P81L/S175G/P530F/E534W/Q550I/R606M +++ 2511/2512Q33H/D130Y/P530F/E534W/Q550I + 2513/2514Q33H/L58M/E59A/I480P/P530F/E534W/Q550I ++ 2515/2516E59A/H154A/P530F/E534W/Q550I + 2517/2518Q33H/W79H/P81L/S175G/P530F/E534W/G603Q + 2519/2520Q33H/L58M/H154A/1480P/E534W/Q550I/G603A/R606M ++ 2521/2522Q33H/P81G/D130Y/1480V/P530F/E534W/Q550I ++ 2523/2524Q33H/W79H/P81G/S175G/P530F/E534W + 2525/2526P81G/I480V/P530F/E534W/Q550I + 2527/2528Q33H/E59A/I480P/P530F/E534W/Q550I/R606M +++ 2529/2530 Q33H/H154A/E534W +2531/2532 W79H/P81I/I480P/P530F/E534W/Q550I/G603E/R606M/G652R ++2533/2534 Q33H/W79H/H154A/I480P/P530F/E534W/Q550I ++ 2535/2536Q33H/P530F/E534W/Q550I + 2537/2538D130Y/I480V/P530F/E534W/Q550I/G603Q/R606M + 2539/2540Q33H/P81I/S175G/P530F/E534W/F542W/Q550I/G652R +++ ^(a)Levels ofincreased production were determined relative to the referencepolypeptide of SEQ ID NO: 2432, and defined as follows: “+” = productionat least 1.2-fold, but less than 5.2-fold; “++” = at least 5.2-fold, butless than 30-fold; and “+++” = at least 30-fold increased production,relative to the reference polypeptide.

TABLE 39.2 SUS Round 13 Saturation Mutagenesis Variants and RebD LevelsAmino Acid Differences SEQ ID NO: (Relative to SEQ ID Increased (nt/aa)NO: 2432) RebD^(a) 2541/2542 L561I +++ 2543/2544 D42H + 2545/2546V267I + 2547/2548 T410S ++ 2549/2550 F70R ++ 2551/2552 F70V + 2553/2554T199A ++ 2555/2556 D42S ++ 2557/2558 A380T + 2559/2560 G758R + 2561/2562A25G + 2563/2564 A25E ++ 2565/2566 D42T +++ 2567/2568 F70S + 2569/2570S265A + 2571/2572 F77W ++ 2573/2574 M75T + 2575/2576 F77L ++ 2577/2578M75W + 2579/2580 F70H + 2581/2582 A25L + 2583/2584 L561V +++ 2585/2586Y106W +++ 2587/2588 F70N +++ 2589/2590 A642V + 2591/2592 G758Q ++2593/2594 S265Q + ^(a)Levels of increased production were determinedrelative to the reference polypeptide of SEQ ID NO: 2432, and defined asfollows: “+” = production at least that of the reference polypeptide,but less than 1.08-fold; “++” = at least 1.08-fold, but less than1.16-fold; and “+++” = at least 1.16-fold increased production, relativeto the reference polypeptide.Shake Flask Powder Characterization Assay and Analysis for GlucosylTransfer from Sucrose to Rebaudioside A

An experiment was performed to characterize the activity of theengineered round 13 SUS variants on sucrose and ADP to facilitate theformation of rebaudioside D from rebaudioside A. Shake flask powder(SFP) was made up to 0.03-1 g/L in potassium phosphate buffer, pH6, andan aliquot was pre-incubated at 73° C. in a thermocycler for 15 minutes.Then, 10 μLof these SFP dilutions, either pre-incubated or notpre-incubated, were added to a 100 μL total reaction volume containing50 mM potassium phosphate buffer, pH6, 15 mM rebaudioside A (>97%purity), 37.5 mM sucrose, 9 mM fructose, 0.2m ADP, and 0.5 g/L GT SEQ IDNO: 4550. The reactions were performed at 55° C. for SFP samples thatwere not pre-incubated and at 60° C. for pre-incubated SFP in aThermotron® titre-plate shaker with 300 RPM shaking for 4h. The reactionwas solubilized by diluting 40× into water, quenched by diluting 5× intoacetonitrile with 0.2% formic acid, and precipitated by centrifugation.The supernatant was diluted 3.3× in water and analyzed for steviolglycosides by RapidFire SPE-MS/MS with the instrument and parametersdescribed in Table 31.1. A further thermostability characterization wasconducted with the clarified shake flask lysates prior to lyophilizationas follows: lysates were diluted 400× in buffer and incubated in athermocycler at a gradient of 62-78° C. for 16.7 h. To determine thepercent activity remaining, the pre-incubated lysates were assayed asdescribed above with either stevioside or rebaudioside D and 4hincubation at 60° C. The percent activity remaining was expressed asactivity at high temperature divided by activity at lowest pre-incubatedtemperature. All 8 of the variants listed in Table 39.3 had higheractivities than SEQ ID NO: 2432 under at least one condition, and fivewere improved under all conditions. The variant with the mutations Q33H,L47P, E59A, P81L, S175G, P530F, E534W, Q5501, and R606M (SEQ ID NO:2510), which was improved under all conditions relative to SEQ IDNO:2432, was selected as the best enzyme for the catalysis of therecycling reaction transferring a glucose from sucrose to ADP.

TABLE 39.3 SUS Round 13 SFP Variants and RebD Levels In- In- % SEQ IDcreased creased Activity NO: Amino Acid Differences RebD, RebD, Re-(nt/aa) (Relative to SEQ ID NO: 2432) 55° C.^(a) 60° C.^(a) maining^(b)2507/2508 Q33H/L58M/E59A/P81G/ − +++ +++ D130Y/I480V/P530F/E534W/Q550I/G652K 2509/2510 Q33H/L47P/E59A/P81L/S175G/ + ++ ++P530F/E534W/Q550I/R606M 2513/2514 Q33H/L58M/E59A/I480P/ + + +P530F/E534W/Q550I 2519/2520 Q33H/L58M/H154A/I480P/ + − +E534W/Q550I/G603A/R606M 2521/2522 Q33H/P81G/D130Y/I480V/ − + ++P530F/E534W/Q550I 2527/2528 Q33H/E59A/I480P/P530F/ + ++ +E534W/Q550I/R606M 2533/2534 Q33H/VV79H/H154A/I480P/ + + +++P530F/E534W/Q550I 2539/2540 Q33H/P81I/S175G/P530F/ + +++ ++E534W/F542W/Q550I/G652R ^(a)Levels of increased production weredetermined relative to the reference polypeptide of SEQ ID NO: 2432, anddefined as follows: “−” = production less than that of the referencepolypeptide; “+” = production at least that of the referencepolypeptide, but less than 1.6-fold; “++” = production at least1.6-fold, but less than 3.7-fold; and “+++” = production at least3.7-fold increased, relative to that of the reference polypeptide.^(b)The percent of activity remaining for each variant was determinedfollowing 16.7 h pre-incubation at 66.5° C., relative to the productionof each variant following pre-incubation at 62° C., and are defined asfollows: “−” = less than 40% of activity remained followingpre-incubation at 62° C.; “+” = at least 40% activity, but less than 60%activity remained; “++” = at least 60% activity, but less than 70%activity remained; and “+++” = at least 70% activity remained.

Example 40 Sucrose Synthase Variants of SEQ ID NO: 2510

Directed evolution of the sucrose synthase encoded by SEQ ID NO: 2509was continued by constructing libraries of variant genes in whichmutations associated with improved production in earlier rounds ofevolution were recombined and in which certain structural features weresubjected to saturation mutagenesis. These libraries were then plated,grown, and screened using the high-throughput (HTP) assay describedbelow, to provide a fourteenth round (“Round 14”) of 164 engineered SuSvariant polypeptides with increased activity toward the generation ofADP-glucose.

HTP Coupled Assay for Glucose Transfer from Sucrose to ADP toRebaudioside A60 to Form Rebaudioside M

Libraries were screened using the following HTP enzyme-coupled assay.Pelleted E. coli cultures were lysed with 400 μL of Tris-HCl, pH 7.5,with 1 mM magnesium sulfate and 0.5 mg/mL lysozyme and polymyxin Bsulfate (PMBS) and cleared by centrifugation. Lysate was diluted 35×into potassium phosphate buffer, pH 6.0, with 14.5 g/L RebA60 andpre-incubated for 15 minutes at 73° C. Then, 10 μL diluted pre-incubatedSuS lysate, 0.08 g/L β1,2GT SFP SEQ ID NO: 4550, and 0.2 g/L β1,3GT SFPSEQ ID NO: 6864 were used in 100 μL reaction volume with 20 g/L RebA60,0.1 g/L ADP (Amresco, ultra pure grade), 40 g/L sucrose (cane sugar),and 9.6 g/L fructose. The following reaction conditions were used: 50 mMpotassium phosphate buffer, pH 6, 60° C. in a Thermotron® titre-plateshaker with 300 RPM shaking for 16-18 h. The reaction described abovewas solubilized by diluting 40× in water, quenched by diluting 5× inacetonitrile with 0.2% formic acid, and precipitated by centrifugation.The supernatant was diluted 15× in water and steviol glycoside productswere detected by RapidFire SPE-MS/MS with the instrument and parametersdescribed in Table 31.1. After analysis, the engineered SuS variantpolypeptides that showed improved activity coupled with a GT onrebaudioside A were identified and are listed in Tables 40.1 and 40.2.Shake-flask scale cultures were grown for protein characterization asdescribed in Example 1 for variants with the amino acid mutations shownin Table 40.3.

TABLE 40.1 SUS Round 14 Combinatorial Variants and RebM Levels SEQ IDNO: Amino Acid Differences Increased (nt/aa) (Relative to SEQ ID NO:2510) RebM^(a) 7437/7438 D259G/Q487I/F684M/L688G + 7439/7440A41K/R71Q/S532Y/F684H +++ 7441/7442 A41K/G485A/Q487K +++ 7443/7444A41K/I91C/G485S + 7445/7446 S532Y ++ 7447/7448 G112W + 7449/7450A41K/G485A/S532Y/L688G + 7451/7452 F684H ++ 7453/7454A41K/G112Q/Q487I/F684H +++ 7455/7456 G112Q ++ 7457/7458G485A/F684H/L688G + 7459/7460 A41K/R71Q + 7461/7462 A41K/D259G/S532Y +7463/7464 A41K/G485S/Q487I/F684M/L688G + 7465/7466 A41K/G112W +7467/7468 G112Q/D259G/S532Y/F684M/L688G + 7469/7470 A41K/S532Y ++7471/7472 A41K/R71Q/G112Q/D259G/G485A/L688Q ++ 7473/7474 G485A/S532Y +++7475/7476 R71Q/F684M/L688A + 7477/7478A41K/D259G/G485A/Q487R/F684H/L688A ++ 7479/7480 S532Y/F684H/L688Q +7481/7482 A41K ++ 7483/7484 G485S/Q487I/F684H/L688Q + 7485/7486A41K/G485A/Q487R ++ 7487/7488 G112Q/G485S/F684H/L688G + 7489/7490D259G/S532Y + 7491/7492 A41K/G112Q/F684H/L688A + 7493/7494 A41K/G485A ++7495/7496 A41K/R71Q/Q487R + 7497/7498 A41K/R71Q/F684H + 7499/7500A41K/R71Q/G485A ++ 7501/7502 A41K/D259G/G485A/Q487I + 7503/7504A41K/D259G/G485S + 7505/7506 A41K/G112Q/G485S/F684H ++ 7507/7508R71Q/G485S/F684M/L688Q + 7509/7510 G485A/Q487R + 7511/7512A41K/R71Q/G112Q +++ 7513/7514 G485S/Q487I/F684M + 7515/7516 G485S +++7517/7518 A41K/G84A/D259G/G485A/Q487I + 7519/7520 G485S/Q487K/S532Y +++7521/7522 R71Q/S532Y ++ 7523/7524 A41K/Q487R/F684M ++ 7525/7526G112Q/G485S/F684M/L688G + 7527/7528 G112Q/D259G/F684M/L688Q + 7529/7530A41K/R71Q/D259G/G485A/S532Y ++ 7531/7532 A41K/G112Q/S532Y ++ 7533/7534A41K/F684M/L688A + 7535/7536 G485A ++ 7537/7538 G485S/Q487R/S532Y/F684H++ 7539/7540 D259G/G485S/Q487K/F684H +++ 7541/7542R71Q/G112W/G485S/L688A ++ 7543/7544 R71Q/G112Q/D259G/G485A/Q487R/F684M +7545/7546 A41K/R71Q/S532Y +++ 7547/7548 G112W/D259G +++ 7549/7550A41K/G485A/S532Y +++ 7551/7552 A41K/I91C/G112Q/G485S/Q487K/S532Y/ +F684H 7553/7554 A41K/Q487I/F684M ++ 7555/7556A41K/R71Q/G485A/S532Y/F684H +++ 7557/7558A41K/G112Q/D259G/Q487R/S532Y/F684H +++ 7559/7560A41K/G112Q/D259G/G485S/Q487R ++ 7561/7562 D259G/G485S/S532Y ++ 7563/7564A41K/F684H + 7565/7566 G485S/F684M/L688G + 7567/7568A41K/D259G/G485A/Q487R/S532Y/F684H + 7569/7570 A41K/G485S/F684M/L688A ++7571/7572 A41K/I91C/G112Q/G485S/S532Y/F684H + 7573/7574A41K/R71Q/Q487R/S532Y/F684H +++ 7575/7576 G112Q/G485S/F684H +++7577/7578 A41K/I91C/G112Q/G485S + 7579/7580 G485A/F684M + 7581/7582R44C/G112W/F684H/L688A + 7583/7584 P226V/Q487I/F684M/L688A + 7585/7586A41K/R71Q/G112W/D259G/G485S/Q487R/ ++ F684H/L688Q 7587/7588F684M/L688Q + ^(a)Levels of increased production were determinedrelative to the reference polypeptide of SEQ ID NO: 2510, and defined asfollows: “+” = production at least 1.5-fold, but less than 2.9-fold;“++” = at least 2.9-fold, but less than 4.2-fold; and “+++” = at least4.2-fold increased production, relative to the reference polypeptide.

TABLE 40.2 SUS Round 14 Saturation Mutagenesis Variants and RebM LevelsSEQ ID NO: Amino Acid Differences Increased (nt/aa) (Relative to SEQ IDNO: 2510) RebM^(a) 7589/7590 R136A ++ 7591/7592 R615L ++ 7593/7594H343N + 7595/7596 E45C + 7597/7598 D12C + 7599/7600 D72E + 7601/7602D12V +++ 7603/7604 R478I + 7605/7606 D12S +++ 7607/7608 P48V + 7609/7610L176M ++ 7611/7612 H343A + 7613/7614 E45G + 7615/7616 R136Q ++ 7617/7618D12Q ++ 7619/7620 T205S + 7621/7622 Q95V ++ 7623/7624 H788K ++ 7625/7626E358P + 7627/7628 D29G + 7629/7630 Q7K/D12L ++ 7631/7632 E100K +++7633/7634 L630M +++ 7635/7636 H724S ++ 7637/7638 P47I +++ 7639/7640Q95N + 7641/7642 I55D ++ 7643/7644 P47V + 7645/7646 R615K + 7647/7648R136P +++ 7649/7650 R478H ++ 7651/7652 E45V ++ 7653/7654 D12N +++7655/7656 R44V + 7657/7658 L176R + 7659/7660 R361A + 7661/7662 R44L +7663/7664 R361L + 7665/7666 D29P +++ 7667/7668 R136K + 7669/7670 P440R++ 7671/7672 L176V + 7673/7674 P47C + 7675/7676 E45A +++ 7677/7678 L116A++ 7679/7680 R303V + 7681/7682 Q27R + 7683/7684 R611A +++ 7685/7686 Q95T+++ 7687/7688 E45L ++ 7689/7690 R615C + 7691/7692 D29A ++ 7693/7694P47N + 7695/7696 R178H + 7697/7698 H724G ++ 7699/7700 R136F +++7701/7702 P47T + 7703/7704 P47L +++ 7705/7706 Q95L + 7707/7708 Q95D ++7709/7710 D29L +++ 7711/7712 R361T + 7713/7714 L176T + 7715/7716 E100P++ 7717/7718 E100Q + 7719/7720 S675C +++ 7721/7722 T205R/G485S +7723/7724 V756C ++ 7725/7726 H724K ++ 7727/7728 P47D + 7729/7730 P207K +7731/7732 Q198A + 7733/7734 Y51P + 7735/7736 P48A + 7737/7738 Q198R ++7739/7740 I55S + 7741/7742 R136N ++ 7743/7744 Y51S ++ 7745/7746 Q201S +7747/7748 R139K ++ 7749/7750 E358S +++ 7751/7752 E317I + 7753/7754R208K + 7755/7756 R615M +++ 7757/7758 K280G + 7759/7760 E45S ++7761/7762 L116F +++ 7763/7764 R478Q + ^(a)Levels of increased productionwere determined relative to the reference polypeptide of SEQ ID NO:2510, and defined as follows: “+” = production at least 1.5-fold, butless than 1.92-fold; “++” = at least 1.92-fold, but less than 2.6-fold;and “+++” = at least 2.6-fold increased production, relative to thereference polypeptide.Shake Flask Powder Characterization Assay and Analysis for GlucosylTransfer from Sucrose to Rebaudioside A60 to form Rebaudioside M

An experiment was performed to characterize the activity of theengineered round 14 SUS variants on sucrose and ADP to facilitate theformation of rebaudioside D from rebaudioside A. Shake flask powder(SFP) was made up to 0.03-1 g/Lin 14.5 μL RebA60 in potassium phosphatebuffer, pH 6, and an aliquot was pre-incubated at 73° C. in athermocycler for 15 minutes. Then, 10 μL diluted, pre-incubated SuSlysate, 0.08 g/L β1,2GT SFP SEQ ID NO: 4550, and 0.2 g/L β1,3GT SFP SEQID NO: 6864, were used in 100 μL reaction volume with 20 μL RebA60, 0.1g/L ADP (Amresco, ultra pure grade), 40 g/L sucrose (cane sugar), and9.6 g/L fructose. The following reaction conditions were used: 50 mMpotassium phosphate buffer, pH 6, 60° C. in a Thermotron® titre-plateshaker with 300 RPM shaking for 16-18 h. The reaction described abovewas solubilized by diluting 40× in water, quenched by diluting 5× inacetonitrile with 0.2% formic acid, and precipitated by centrifugation.The supernatant was diluted 15× in water and steviol glycoside productswere detected by RapidFire SPE-MS/MS with the instrument and parametersdescribed in Table 31.1. All 8 of the variants listed in Table 40.3 hadhigher activities than SEQ ID NO: 2510. The variant with the mutationsA41K, G112Q, G485S, and F684H (SEQ ID NO:7506), which was most improvedrelative to SEQ ID NO: 2510, was selected as the best enzyme for thecatalysis of the recycling reaction transferring a glucose from sucroseto ADP.

TABLE 40.3 SUS Round 14 SFP Variants and RebM Levels SEQ ID NO: AminoAcid Differences (nt/aa) (Relative to SEQ ID NO: 2510) IncreasedRebM^(a) 7439/7440 A41K/R71Q/S532Y/F684H +++ 7453/7454A41K/G112Q/Q487I/F684H ++ 7493/7494 A41K/G485A + 7505/7506A41K/G112Q/G485S/F684H +++ 7515/7516 G485S + 7549/7550 A41K/G485A/S532Y++ 7555/7556 A41K/R71Q/G485A/S532Y/F684H + 7575/7576 G112Q/G485S/F684H +^(a)Levels of increased production were determined relative to thereference polypeptide of SEQ ID NO: 2510, and defined as follows: “+” =production at least 2.1-fold, but less than 2.85-fold; “++” = productionat least 2.85-fold, but less than 3.1-fold; and “+++” = production atleast 3.1-fold increased, relative to that of the reference polypeptide.

Example 41 Sucrose Synthase Variants of SEQ ID NO: 7506

Directed evolution of the sucrose synthase encoded by SEQ ID NO: 7505was continued by constructing libraries of variant genes in whichmutations associated with improved production in earlier rounds ofevolution were recombined and in which certain structural features weresubjected to saturation mutagenesis. These libraries were then plated,grown, and screened using the high-throughput (HTP) assay describedbelow, to provide a fifteenth round (“Round 15”) of 56 engineered SuSvariant polypeptides with increased activity toward the generation ofADP-glucose.

HTP Coupled Assay for Glucose Transfer from Sucrose to ADP toRebaudioside A 60 to Form Rebaudioside M

Libraries were screened using the following HTP enzyme-coupled assay.Pelleted E. coli cultures were lysed with 400 μL of Tris-HCl, pH 7.5with 1 mM magnesium sulfate and 0.5 mg/mL lysozyme and polymyxin Bsulfate (PMBS) and cleared by centrifugation. Lysate was diluted 50×into potassium phosphate buffer, pH 6.0, with 14.5 g/L RebA60 andpre-incubated for 15 minutes at 73° C. Then, 10 μL diluted,pre-incubated SuS lysate, 0.08 g/L β1,2GT SFP SEQ ID NO: 4550, and 0.2g/L β1,3GT SFP SEQ ID NO: 6864, were used in 100 μL reaction volume with20 g/L RebA60, 0.1 g/L ADP (Amresco, ultra pure grade), 40 g/L sucrose(cane sugar), and 9.6 g/L fructose. The following reaction conditionswere used: 50 mM potassium phosphate buffer, pH 6, 60° C. in aThermotron® titre-plate shaker with 300 RPM shaking for 16-18 h. Thereaction described above was solubilized by diluting 20× in water,quenched by diluting 5× in acetonitrile with 0.2% formic acid, andprecipitated by centrifugation. The supernatant was diluted 20× in waterand steviol glycoside products were detected by RapidFire SPE-MS/MS withthe instrument and parameters described in Table 31.1. After analysis,the engineered SuS variant polypeptides that showed improved activitycoupled with a GT on rebaudioside A were identified and are listed inTable 41.1. Shake-flask scale cultures were grown for proteincharacterization as described in Example 1 for variants with the aminoacid mutations shown in Table 41.2.

TABLE 41.1 SUS Round 15 Combinatorial Variants and RebM Levels SEQ IDNO: Amino Acid Differences Increased (nt/aa) (Relative to SEQ ID NO:7506) RebM^(a) 8369/8370 D12N/E45A/P47I/Y51P/R136P/R139K/ ++ L630M/G758R8371/8372 D12N/E45A/Y51P/R136P/R139K/L630M/ +++ S675C/V756C/G758Q8373/8374 D12S/E45A/Y51P/L630M/V756C ++ 8375/8376 R136P/R139K/S142N ++8377/8378 D12N/R136Q/S142N +++ 8379/8380D12V/E45A/P47I/R136Q/R139K/S142N/ +++ S675C/G758Q 8381/8382D12V/E45A/P47L +++ 8383/8384 D12V/R136Q/R139K/S142N/V756C/G758Q +++8385/8386 D12S/L630M/V756C ++ 8387/8388 D12N/P48A/Y51P/R136Q/R139K/G758Q+++ 8389/8390 D12V/E45A/P47I/P48A/Y51S/R136Q/ +++ S142N/L630M 8391/8392P47I/Y51S/R136Q/V756C/G758Q ++ 8393/8394T199A/S532Y/S539R/L561I/G652R/H724S + 8395/8396 S532Y/L561I/H724K +8397/8398 Q198R/I480P/L561I ++ 8399/8400 Q198R/T199A/L561V/H724G +++8401/8402 D42T/Q198R/S532Y/L561I/H724K ++ 8403/8404Y106W/T199A/S539R/L561I/G652R ++ 8405/8406 I480V/L561V/G652R ++8407/8408 Q198R/I480V/H724K ++ 8409/8410 D42T/Q198R/S532Y/L561V ++8411/8412 D42T/D259G/I480V/L561V + 8413/8414 Q198R/T199A/I480P/L561I ++8415/8416 S532Y/S539R/L561V + 8417/8418 D42T/Q198R/T199A/I480V/S532Y/+++ S539R/L561V 8419/8420 D42T/I480V/L561I/H724K +++ 8421/8422D42T/I480P/L561I ++ 8423/8424 D42T/L561I ++ 8425/8426 D259G/I480V +8427/8428 I480V/L561V/G652R/H724K/L764R + 8429/8430 I480V/H724K ++8431/8432 Q198R/T199A/I480P/L561V/H724K ++ 8433/8434D42T/D259G/I480V/G652K + 8435/8436 D42T/T199A/I480V/S532Y/L561V +++8437/8438 A25E/A517P + 8439/8440 H154A/R361T/P440R/A517P/G603E/H788K +8441/8442 Q95T/G603E + 8443/8444 A25E/E100K/H154A/R208K/P440R/ +A517P/P705M/H788K 8445/8446 A25E/D29P/R208K/P440R + 8447/8448K280G/P440R/A517P + 8449/8450 A517P + 8451/8452 I55D/A517P/H788K ++8453/8454 I55D/A517P + 8455/8456 I55D + 8457/8458Q8R/A25E/I55D/Q95T/R208K/E358S/ + P440V/A517P/H788K 8459/8460D29P/R208K/R361T/A517P/H788K + 8461/8462 G603E + 8463/8464I55D/T410S/P440R/G603E/H788K + 8465/8466 A642V + 8467/8468F77L/L176M/Q487K/R615M/A642V + 8469/8470 F70N/A642V + 8471/8472 L116F +8473/8474 A380T + 8475/8476 L176T + 8477/8478 F70N + 8479/8480V267I/R611Q/A642V + ^(a)Levels of increased production were determinedrelative to the reference polypeptide of SEQ ID NO: 7506, and defined asfollows: “+” = activity at least 1.07-fold, but less than 1.95-fold;“++” = at least 1.95-fold, but less than 2.4-fold; and “+++” = at least2.4-fold increased production, relative to the reference polypeptide.Shake Flask Powder Characterization Assay and Analysis for GlucosylTransfer from Sucrose to Rebaudioside A60 to form Rebaudioside M

An experiment was performed to characterize the activity of theengineered round 15 SUS variants on sucrose and ADP to facilitate theformation of rebaudioside D from rebaudioside A. Shake flask powder(SFP) was made up to 0.03-1 g/L in 14.5 g/L RebA6 in potassium phosphatebuffer, pH 6, and an aliquot was pre-incubated at 73° C. in athermocycler for 15 minutes. 10 μL diluted, pre-incubated SuS lysate,0.08 g/L β1,2GT SFP SEQ ID NO: 4550, and 0.2 g/L β1,3GT SFP SEQ ID NO:6864, were used in 100 μL reaction volume with 20 g/L RebA60, 0.1 g/LADP (Amresco, ultra pure grade), 40 g/L sucrose (cane sugar), and 9.6g/L fructose. The following reaction conditions were used: 50 mMpotassium phosphate buffer, pH 6, 60° C. in a Thermotron® titre-plateshaker with 300 RPM shaking for 16-18 h. The reaction described abovewas solubilized by diluting 20× in water, quenched by diluting 5× inacetonitrile with 0.2% formic acid, and precipitated by centrifugation.The supernatant was diluted 20× in water and steviol glycoside productswere detected by RapidFire SPE-MS/MS with the instrument and parametersdescribed in Table 31.1. All 8 of the variants listed in Table 41.2 hadhigher activities than SEQ ID NO: 7506. The variant with the mutationsD42T, I480V, L561I, and H724K (SEQ ID NO: 8420), which was most improvedrelative to SEQ ID NO: 7506, was selected as the best enzyme for thecatalysis of the recycling reaction transferring a glucose from sucroseto ADP.

TABLE 41.2 SUS Round 15 SFP Variants and RebM Levels SEQ ID NO: AminoAcid Differences Increased (nt/aa) (Relative to SEQ ID NO: 7506)RebM^(a) 8371/8372 D12N/E45A/Y51P/R136P/R139K/L630M/ + S675C/V756C/G758Q8383/8384 D12V/R136Q/R139K/S142N/V756C/G758Q + 8389/8390D12V/E45A/P47I/P48A/Y51S/R136Q/ ++ S142N/L630M 8399/8400Q198R/T199A/L561V/H724G + 8419/8420 D42T/I480V/L561I/H724K +++ 8439/8440H154A/R361T/P440R/A517P/G603E/H788K +++ 8451/8452 I55D/A517P/H788K ++8475/8476 L176T + ^(a)Levels of increased production were determinedrelative to the reference polypeptide of SEQ ID NO: 7506, and defined asfollows: “+” = production at least 2.1-fold, but less than 2.2-fold;“++” = production at least 2.2-fold, but less than 2.3-fold; and “+++” =production at least 2.3-fold increased, relative to that of thereference polypeptide.

Example 42 Sucrose Synthase Variants of SEQ ID NO: 8420

Directed evolution of the sucrose synthase encoded by SEQ ID NO: 8419was continued by constructing libraries of variant genes in whichmutations associated with improved production in earlier rounds ofevolution were recombined. These libraries were then plated, grown, andscreened using the high-throughput (HTP) assay described below, toprovide a sixteenth round (“Round 16”) of 155 engineered SuS variantpolypeptides with increased activity toward the generation ofADP-glucose.

HTP Coupled Assay for Glucose Transfer from Sucrose to ADP toRebaudioside A 60 to Form Rebaudioside M

Libraries were screened using the following HTP enzyme-coupled assay.Pelleted E. coli cultures were lysed with 400 μL of Tris-HCl, pH 7.5with 1 mM magnesium sulfate and 0.5 mg/mL lysozyme and polymyxin Bsulfate (PMBS) and cleared by centrifugation. Lysate was diluted 30×into potassium phosphate buffer, pH 6.0, with 14.5 g/L RebA60 andpre-incubated for 1 hour at 75° C. Then, 10 μL diluted, pre-incubatedSuS lysate, 0.08 g/L β1,2GT SFP SEQ ID NO: 7784, and 0.2 g/L β1,3GT SFPSEQ ID NO: 8088, were used in 100 μL reaction volume with 20 g/L RebA60,0.025 g/L ADP (Amresco, ultra pure grade), 40 g/L sucrose (cane sugar),and 9.6 g/L fructose. The following reaction conditions were used: 50 mMpotassium phosphate buffer, pH 6, 60° C. in a Thermotron® titre-plateshaker with 300 RPM shaking for 16-18 h. The reaction described abovewas solubilized by diluting 20× in water, quenched by diluting 5× inacetonitrile with 0.2% formic acid, and precipitated by centrifugation.The supernatant was diluted 20× in water and steviol glycoside productswere detected by RapidFire SPE-MS/MS with the instrument and parametersdescribed in Table 31.1. After analysis, the engineered SuS variantpolypeptides that showed improved activity coupled with a GT onrebaudioside A were identified and are listed in Table 42.1. Shake-flaskscale cultures were grown for protein characterization as described inExample 1 for variants with the amino acid mutations shown in Table42.2.

TABLE 42.1 SUS Round 16 Combinatorial Variants and RebM Levels SEQ IDNO: Amino Acid Differences Increased (nt/aa) (Relative to SEQ ID NO:8420) RebM^(a) 8797/8798 D12S/E45A/L176T/G603Q/L630M/ + A642V/V756C8799/8800 D12N/T199A/R208K/A517P/A642V + 8801/8802D12N/E45A/R136Q/R208K/A517P/ +++ L630M/V756C 8803/8804D12S/R139K/G603Q/V756C + 8805/8806 R136Q/R139K/G603Q + 8807/8808R136Q/R139K/A517P/G603Q/V756C +++ 8809/8810D12S/R136Q/T199A/R208K/L630M/A642V ++ 8811/8812D12S/R136Q/R139K/R208K/A517P ++ 8813/8814D12N/E45A/R136Q/R139K/A642V/V756C ++ 8815/8816D12S/R139K/L176T/L630M/V756C + 8817/8818D12S/R136Q/A517P/G603Q/L630M/A642V ++ 8819/8820D12S/R136Q/R139K/L176T/V756C + 8821/8822 D12N/E45A/R136Q/A517P/L630M/+++ A642V/V756C 8823/8824 D12N/E45A/R136Q/G603E/V756C + 8825/8826D12S/E45A/R136Q/R139K/R208K/G603E/ ++ L630M/V756C 8827/8828D12S/R136Q/L176T/R208K/A517P/G603E/ +++ L630M/V756C 8829/8830D12N/L176T/A517P/V756C + 8831/8832 D12S/R136Q/R208K/G603Q/A642V +8833/8834 D12N/R136Q/R139K/G603Q/A642V/V756C +++ 8835/8836D12S/R136Q/R139K/G603Q/L630M/V756C +++ 8837/8838D12S/R136Q/R139K/L176T/A517P/ +++ G603Q/L630M 8839/8840D12N/R136Q/R139K/L630M + 8841/8842 D12S/R136Q/R139K/A517P/L630M ++8843/8844 D12N/R136Q/T199A/A517P/V756C + 8845/8846D12S/R136Q/R139K/T199A/R208K/A517P/ +++ G603E/V756C 8847/8848D12N/E45A/R136Q/R139K/T199A/A517P/ ++ G603E 8849/8850D12N/A517P/G603E/V756C + 8851/8852 D12N/R136Q/G603Q/L630M/V756C ++8853/8854 D12N/R136Q/R139K/A642V/V756C + 8855/8856D12S/R136Q/R139K/L630M/A642V/V756C +++ 8857/8858D12N/E45A/R139K/T199A/R208K/G603E + 8859/8860T199A/A517P/G603Q/L630M/V756C + 8861/8862 R136Q/R139K/L176T/A517P/L630M/+++ A642V/V756C 8863/8864 R136Q/R139K/L176T/T199A/R208K/A517P/ ++L630M/A642V 8865/8866 D12S/R136Q/L176T/A517P/A642V + 8867/8868D12N/R139K/G603Q/V756C + 8869/8870 D12N/R136Q/G603E/A642V ++ 8871/8872D12N/E45A/R136Q/R139K/A517P/L630M/ +++ A642V/V756C 8873/8874D12S/R136Q/L630M + 8875/8876 D12N/R136Q/R139K/A517P/V756C ++ 8877/8878R136Q/R139K/A517P/G603E/A642V/V756C +++ 8879/8880D12S/R136Q/R139K/G603Q/L630M/A642V ++ 8881/8882 D12N/G603Q/L630M/V756C +8883/8884 D12S/A517P/G603Q/L630M/A642V/V756C ++ 8885/8886D12N/E45A/R136Q/R139K/G603E/V756C ++ 8887/8888D12N/R136Q/R139K/A517P/L630M/A642V ++ 8889/8890D12S/R136Q/R139K/L630M/V756C ++ 8891/8892 D12N/Q95T/A517P/L630M/V756C +8893/8894 D12S/Q95T/R139K/A517P/L630M/V756C ++ 8895/8896D12N/E45A/T199A/R208K/A517P/G603E/ + L630M/V756C 8897/8898D12N/R136Q/L176T/G603Q/V756C + 8899/8900D12S/R136Q/G603Q/L630M/A642V/V756C ++ 8901/8902D12N/T199A/L630M/A642V/V756C + 8903/8904 D12N/L630M/A642V/V756C +8905/8906 D12S/R136Q/A517P/G603Q/L630M/V756C ++ 8907/8908D12N/E45A/R136Q/R139K/A517P/G603E/ +++ V756C 8909/8910D12S/R136Q/R139K/A517P/G603Q/L630M/ +++ A642V/V756C 8911/8912D12N/Q95T/R136Q/R139K/A517P/G603Q/ ++ V756C 8913/8914D12N/R208K/L630M/V756C + 8915/8916 D12N/R136Q/R139K/G603E/L630M +++8917/8918 D12N/R136Q/A517P/L630M +++ 8919/8920R136Q/R139K/A517P/G603Q/L630M/ +++ A642V/V756C 8921/8922D12S/R136Q/A517P/A642V/V756C +++ 8923/8924 R139K/A517P/L630M/V756C ++8925/8926 D12S/R139K/A517P/L630M/A642V/V756C ++ 8927/8928A517P/G603E/L630M/A642V/V756C + 8929/8930 D12N/L176T/A517P/G603E/L630M +8931/8932 D12S/A517P/A642V/V756C ++ 8933/8934 D12S/E45A/Q95T/R136Q/V756C++ 8935/8936 D12N/R139K/A517P/G603Q/A642V/V756C +++ 8937/8938R136Q/A517P/V756C + 8939/8940 D12N/G603Q/A642V/V756C ++ 8941/8942D12S/T199A/V756C + 8943/8944 D12S/E45A/R139K/V756C + 8945/8946D12S/L176T/G603Q/L630M/V756C ++ 8947/8948 D12S/R139K/R208K/A642V +8949/8950 D12N/E45A/Q95T/R136Q/R139K/T199A/ +++ A517P/L630M/V756C8951/8952 D12S/R136Q/R139K/A517P/G603E/V756C +++ 8953/8954D12N/R136Q/T199A/R208K/A517P/G603E/ +++ A642V/V756C 8955/8956R136Q/R139K/R208K/A517P/L630M/V756C +++ 8957/8958R136Q/R139K/T199A/A517P/G603E/V756C +++ 8959/8960 D12S/L630M/V756C +8961/8962 D12S/G603Q/L630M/V756C ++ 8963/8964 R136Q/R139K/A517P/V756C ++8965/8966 D12N/Q95T/R139K/L630M/A642V + 8967/8968 D12N/R136Q + 8969/8970D12N/R139K/T199A/A517P/L630M/V756C +++ 8971/8972 R139K/A642V/V756C +8973/8974 D12S/R136Q/R139K/A517P/G603E/ +++ L630M/V756C 8975/8976D12S/R139K/L176T/A517P/G603Q/ +++ L630M/V756C 8977/8978D12N/A517P/L630M/A642V/V756C + 8979/8980 D12S/R136Q/A517P/L630M/A642V +8981/8982 D12N/R139K/L630M/A642V/V756C ++ 8983/8984D12N/R136Q/R139K/L176T/A517P/ +++ G603Q/V756C 8985/8986R136Q/R139K/L630M/A642V/V756C ++ 8987/8988 D12S/R139K/A517P/A642V +8989/8990 D12N/R136Q/G603Q/V756C ++ 8991/8992D12N/Q95T/T199A/A517P/A642V + 8993/8994 D12N/R139K/G603E/A642V/V756C ++8995/8996 R139K/A517P/G603Q/V756C + 8997/8998D12N/R136Q/R139K/L176T/L630M/V756C +++ 8999/9000D12S/E45A/R208K/A517P/G603E/A642V/ + V756C 9001/9002D12N/R136Q/G603E/A642V/V756C ++ 9003/9004 D12N/A517P/G603Q/L630M/V756C +9005/9006 R139K/T199A/A517P/A642V + 9007/9008 G603Q/V756C + 9009/9010D12N/R139K/L630M/V756C ++ 9011/9012 D12S/R136Q/A517P/V756C ++ 9013/9014D12S/E45A/R136Q/L176T/A517P/G603E/ + L630M/A642V 9015/9016 R136Q/A642V +9017/9018 D12S/A517P/L630M/A642V/V756C ++ 9019/9020D12N/R136Q/R139K/L176T/A642V ++ 9021/9022 D12N/R136Q/R208K + 9023/9024D12S/R136Q/G603E/V756C ++ 9025/9026 D12N/R139K/T199A/R208K/A642V +9027/9028 D12N/R208K/A517P/G603E/H623N/ + L630M/A642V 9029/9030A517P/L630M/V756C + 9031/9032 D12S/R136Q/A517P/A642V + 9033/9034D12N/E45A/R136Q/L630M/A642V + 9035/9036D12S/R136Q/R139K/A517P/G603E/L630M/ +++ A642V/V756C 9037/9038D12N/G603Q/V756C + 9039/9040 D12S/Q95T/L630M/V756C + 9041/9042R136Q/A642V/V756C + 9043/9044 D12N/R139K/L630M/A642V + 9045/9046D12N/L176T/A517P/L630M/A642V/V756C ++ 9047/9048 R136Q/V756C + 9049/9050D12S/E45A/R136Q/R139K/A517P/V756C ++ 9051/9052 D12N/G603E/A642V/V756C +9053/9054 R136Q/G603Q/V756C + 9055/9056 D12N/R136Q/R139K/A517P ++9057/9058 R208K/A517P/L630M/A642V/V756C + 9059/9060 R136Q/L630M/A642V +9061/9062 D12N/R136Q/R139K/T199A/R208K/A517P/ ++ L630M/A642V/V756C9063/9064 R139K/R208K/A517P/L630M/A642V/V756C + 9065/9066R136Q/L630M/V756C + 9067/9068 D12S/E45A/R139K/A517P/V756C + 9069/9070R136Q/R139K/R208K/G603Q/L630M/V756C ++ 9071/9072D12N/T199A/A642V/V756C + 9073/9074 D12S/R208K/G603Q/L630M/V756C +9075/9076 D12N/Q95T/R139K/A517P/A642V + 9077/9078D12S/L176T/G603E/V756C + 9079/9080 D12N/R136Q/R139K/A517P/L630M/ ++A642V/V756C 9081/9082 D12S/R136Q/R139K/A517P/G603Q/V756C ++ 9083/9084D12S/R136Q/R139K/A517P/L630M/A642V ++ 9085/9086 D12S/R136Q/L630M/V756C +9087/9088 D12N/R139K/R208K/A517P + 9089/9090D12N/E45A/R139K/A517P/V756C + 9091/9092D12S/E45A/R136Q/A517P/G603Q/A642V/ + V756C/A789V 9093/9094D12S/E45A/R139K/L176T/R208K/A517P/ + G603E/L630M 9095/9096D12N/R136Q/G603Q/A642V/V756C ++ 9097/9098R139K/T199A/R208K/A517P/L630M/V756C + 9099/9100 G603E/L630M/V756C +9101/9102 A25E/L176M/Q198R/S532Y/S539R + 9103/9104H154A/S532Y/G652R/H788K + 9105/9106 L116F/S142N/Q198R/Y434H/P440R +^(a)Levels of increased production were determined relative to thereference polypeptide of SEQ ID NO: 8420, and defined as follows: “+” =production at least 7.8-fold, but less than 16-fold; “++” = at least16-fold, but less than 22.3-fold; and “+++” = at least 22.3-foldincreased production, relative to the reference polypeptide.Shake Flask Powder Characterization Assay and Analysis for GlucosylTransfer from Sucrose to Rebaudioside A 60 to form Rebaudioside M

An experiment was performed to characterize the activity of theengineered round 16 SUS variants on sucrose and ADP to facilitate theformation of rebaudioside M from rebaudioside A. Shake flask powder(SFP) was made up to 0.03-1 g/Lin 4.5 g/L RebA6 in potassium phosphatebuffer, pH 6, and an aliquot was pre-incubated at 75° C. for 1 hour. 10μL diluted, either pre-incubated or not pre-incubated SuS lysate, 0.08g/L β1,2GT SFP SEQ ID NO: 7784, and 0.2 g/L β1,3GT SFP SEQ ID NO: 8088,were used in 100 μL reaction volume with 20 μL RebA60, 0.025 g/L ADP(Amresco, ultra pure grade), 40 g/L sucrose (cane sugar), and 9.6 g/Lfructose. The following reaction conditions were used: 50 mM potassiumphosphate buffer, pH 6, 60° C. in a Thermotron® titre-plate shaker with300 RPM shaking for 16-18 h. The reaction described above wassolubilized by diluting 20× inwater, quenched by diluting 5× inacetonitrile with 0.2% formic acid, and precipitated by centrifugation.The supernatant was diluted 20× in water and steviol glycoside productswere detected by RapidFire SPE-MS/MS with the instrument and parametersdescribed in Table 31.1. All 8 of the variants listed in Table 42.2 hadhigher production following preincubation than SEQ ID NO:8420. Thevariant with the mutations D12S, R136Q, R139K, A517P, G603Q, L630M,A642V, and V756C (SEQ ID NO: 8910), which was most improved relative toSEQ ID NO:8420, was selected as the best enzyme for the catalysis of therecycling reaction transferring a glucose from sucrose to ADP.

TABLE 42.2 SUS Round 16 SFP Variants and RebM Levels Amino AcidDifferences Increased RebM Increased RebM SEQ ID NO: (Relative towithout pre- with 75° C. pre- (nt/aa) SEQ ID NO: 8420) incubation^(a)incubation^(a) 8827/8828 D12S/R136Q/L176T/R208K/A517P/G60 + ++3E/L630M/V756C 8909/8910 D12S/R136Q/R139K/A517P/G603Q/L630M/ + +++A642V/V756C 8919/8920 R136Q/R139K/A517P/G603Q/L630M/ + +++ A642V/V756C8953/8954 D12N/R136Q/T199A/R208K/A517P/ − ++ G603E/A642V/V756C 9035/9036D12S/R136Q/R139K/A517P/G603E/ − +++ L630M/A642V/V756C 9079/9080D12N/R136Q/R139K/A517P/L630M/ + +++ A642V/V756C 9101/9102A25E/L176M/Q198R/S532Y/S539R − ++ 9105/9106L116F/S142N/Q198R/Y434H/P440R − ++ ^(a)Levels of increased productionwere determined relative to the reference polypeptide of SEQ ID NO:8420, and defined as follows: “−” = production less than that of thereference polypeptide; “+” = production at least that of the referencepolypeptide, but less than 3-fold; “++” = production at least 3-fold,but less than 13-fold; and “+++” = production at least 13-foldincreased, relative to that of the reference polypeptide.

Example 43 Beta-1,2-ADP-Glycosyltransferase Variants of SEQ ID NO: 1054

In this Example, experiments for evolution and screening of GTpolypeptides (β1,2GT) derived from SEQ ID NO: 1054, for improvedglucosylation of steviol glycosides using ADP-glucose are described.Directed evolution of the GT encoded by SEQ ID NO: 1053 was carried outby constructing libraries in which mutations associated with improvedactivity in previous rounds were recombined. These libraries were thenplated, grown, and screened using the high-throughput (HTP) assaydescribed below to provide a fifth round (“Round 5”) of 26 engineered GTvariant polypeptides with glucosyltransferase activity towardADP-glucose and steviol glycosides.

HTP Assay for Glucose Transfer from ADP-Glucose to Rebaudioside A

Assays were performed on 96-well plates of cleared E. coli culturelysates expressing SEQ ID NO: 1053 variants. Lysis buffer volume was 400μL, and the lysate was diluted 4-fold. Assays were conducted with 10 μLdiluted lysate in 100 μL reaction volume with substrate loading of 1 mMrebaudioside A (>97% purity) and co-substrate loading of 1 mMADP-glucose (Sigma, >93% purity). The following reaction conditions wereused: 50 mM potassium phosphate buffer, pH 7, 3 mM MgCl₂, 50° C. in aThermotron® titre-plate shaker with 300 RPM shaking for 1 h. Thereactions were diluted 1:5 in water and then quenched by adding 25 μL ofthe diluted assay to 75 μL acetonitrile with 0.2% formic acid. Theresulting mixture was precipitated by centrifugation at 4° C. for 10 m.The supernatants were diluted 1:5 in water and analyzed by RapidFireSPE-MS/MS as described in Table 31.1. Glycosyltransferase variantpolypeptides that produced rebaudioside D from rebaudioside A withADP-glucose were identified, and the engineered polypeptides are listedin Table 43.1. Shake-flask scale cultures were grown, lysed, andlyophilized to powder as described in Example 31 for the variants listedin Table 43.2.

TABLE 43.1 β1,2GT Round 5 Variants and RebD Levels SEQ ID NO: Amino AcidDifferences Increased (nt/aa) (Relative to SEQ ID NO: 1054) RebD^(a)2595/2596 E24L/N162R/P175S/M201G/D275Q/I316V + 2597/2598E24L/W226V/P330Q/L351M/K403R + 2599/2600E24L/N162R/E198P/M201G/T211E/W226V/ + L323V/L351M 2601/2602E24L/P175S/M201G/D275Q/I316V/L351M + 2603/2604E24L/M201G/W226V/G253D/L402I/ ++ K403R/I406M 2605/2606P175S/E198P/M201G/T211E/W226V/T260V/ +++ S264A/L323V/L402I/I406M2607/2608 E24L/P175S/M201G/I316V + 2609/2610E24L/P175S/T211E/I316V/P330Q + 2611/2612 E24L/P175S/T211E/G253D/I316V ++2613/2614 E24L/E198P/M201G/T211E ++ 2615/2616E24L/P175S/W226V/L323V/L351M + 2617/2618 E24L/E198P/M201G + 2619/2620E24L/M201G/T211E/G253D/L323V/L351M/ + Q366H/D389E/L402I/K403R 2621/2622E24L/P175S/T211E/C220L/T260V/D275Q/ + P330Q/D389E 2623/2624E24L/E198P/M201G/T211E/C220L/T260V +++ 2625/2626P175S/E198P/W226V/T260V/L351M/L402I/ ++ K403R/I406M 2627/2628E24L/E198P/M201G/W226V/P330Q/D389E ++ 2629/2630E24L/N32S/P175S/W226V/G253D/D275Q/ + I316V 2631/2632E24L/N32S/P175S/T211E/T260V/P330Q/ + K403R/I406M 2633/2634E24L/N162R/M201G/G253D/S264A/L351M/ ++ L402I/I406M 2635/2636E24L/E198P/M201G/L351M ++ 2637/2638 E24L/N162R/E198P/M201G/W226V/L351M+++ 2639/2640 E24L/N162R/P175S/E198P/T211E/W226V/ +D275Q/I316V/L323V/D389E 2641/2642 E24L/P175S/E198P/T211E/W226V/G253D/+++ I316V 2643/2644 E24L/E198P/M201G/C220L/D275Q/D389E/ ++ L402I/I406M2645/2646 E24L/N32S/M201G/W226V/T260V/S264A/ +++ P330Q/L402I/I406M^(a)Levels of increased production were determined relative to thereference polypeptide of SEQ ID NO: 1054, and defined as follows: “+” =production at least 4.1-fold, but less than 4.58-fold; “++” = at least4.58-fold, but less than 5.17-fold; and “+++” at least 5.17-foldincreased production, as compared to the reference polypeptide.Shake Flask Powder Characterization Assay and Analysis for GlucosylTransfer from ADP-Glucose to Rebaudioside A

A shake flask powder loading dose response experiment was performed tocharacterize activity of the engineered round 5 variants on rebaudiosideA. Levels of 0.006-0.2 g/L shake flask powder (SFP) were added to a 100μL total reaction volume containing 50 mM potassium phosphate buffer, pH7, 3 mM MgCl₂, 1 mM rebaudioside A, and 1 mM ADP-glucose. The reactionwas performed at 50° C. in a Thermotron® titre-plate shaker at 300 RPMfor 1 h. The reaction was diluted 1:5 in water and then quenched byadding 25 μL of the diluted assay to 75 μL acetonitrile with 0.2% formicacid and precipitated by centrifugation at 4° C. for 10 m. Thesupernatant was diluted 1:5 in water and analyzed for steviol glycosidesby RapidFire SPE-MS/MS. The activities of the round 5 variants onrebaudioside A at 0.05 g/L SFP loading are listed in Table 43.2. All 6of the variants listed in Table 43.2 had higher activities than SEQ IDNO: 1054. The variant with the mutations E24L, N162R, E198P, M201G,T211E, W226V, L323V, and L351M (SEQ ID NO: 2600) and its encodingpolynucleotide (SEQ ID NO: 2599) were selected for further directedevolution for the glucosylation of rebaudioside A.

TABLE 43.2 β1,2GT Round 5 SFP Variants and RebD Levels SEQ ID NO: AminoAcid Differences Increased (nt/aa) (Relative to SEQ ID NO: 1054)RebD^(a) 2595/2596 E24L/N162R/P175S/M201G/D275Q/ ++ I316V 2599/2600E24L/N162R/E198P/M201G/T211E/ +++ W226V/L323V/L351M 2605/2606P175S/E198P/M201G/T211E/VV226V/ +++ T260V/S264A/L323V/L402I/I406M2623/2624 E24L/E198P/M201G/T211E/C220L/T260V + 2633/2634E24L/N162R/M201G/G253D/S264A/ + L351M/L402I/I406M 2637/2638E24L/N162R/E198P/M201G/W226V/L351M ++ ^(a)Levels of increased productionwere determined relative to the reference polypeptide of SEQ ID NO:1054, and defined as follows: “+” = production at least 8.75-fold, butless than 10-fold; “++” = at least 10-fold, but less than 11-fold; and“+++” at least 11-fold increased production, relative to the referencepolypeptide.

Example 44 Glycosyltransferase Variants of SEQ ID NO: 1002 forGlucosylation of Rebaudioside I to Rebaudioside M

In this Example, experiments for evolution and screening of GTpolypeptides derived from SEQ ID NO: 1002 for improved glucosylation ofsteviol glycosides are described. Directed evolution of the GT encodedby SEQ ID NO: 1001 was carried out by constructing libraries of variantgenes in which mutations associated with improved glycosyltransferaseactivity toward rebaudioside I in previous rounds were recombined. Theselibraries were then plated, grown, and screened using thehigh-throughput (HTP) assay described below to provide a round (Round 5RebI) of engineered GT variant polypeptides with glucosyltransferaseactivity toward UDP-glucose and rebaudioside I.

HTP Assay for Glucose/Transfer from UDP-Glucose to Rebaudioside I

Cells were lysed with 250 lysis buffer. Assays were performed on 96-wellplates of cleared E. coli culture lysates expressing SEQ ID NO: 1002variants with lysate loading of 25 μL lysate in 100 μL reactions andwith substrate loading of 1 mM rebaudioside I and co-substrate loadingof 1 mM UDP-glucose (Sigma, >98% purity). The following reactionconditions were used: 50 mM KPhos buffer, pH7, 3 mM MgCl₂, 40° C. in aThermotron® titre-plate shaker with 300 RPM shaking for 18h. Thereactions were quenched by adding 10 μL of the assay to 90 μLacetonitrile with 0.2% formic acid. The resulting mixture wasprecipitated by centrifugation at 4° C. for 10 m. The supernatants werediluted 1:5 in water and analyzed by LC-MS/MS as described in Table44.1. Glycosyltransferase variant polypeptides that producedrebaudioside M from rebaudioside I with UDP-glucose, at greaterquantities than SEQ ID NO: 1002 were identified. The engineeredpolypeptides are listed in Table 44.2.

TABLE 44.1 HPLC-MS/MS Analysis of Steviol Glycosides for Rebaudioside IProducts. Instrument Agilent HPLC 1200 series, Sciex 4000 QTrap ColumnPoroshell 120 EC C18 50 × 3.0 mm, 2.7 μm with Poroshell 120 EC C18 5 ×3.0, 2.7 μm guard column (Agilent Technologies) Mobile phase Gradient(A: 0.1% formic acid in water, B: 0.1% formic acid in methanol) Time (m)% B 0 40 0.50 53 5.00 53 5.50 70 7.5 70 8.00 95 8.50 95 8.51 60 9.20 40Flow rate 0.8 mL/m Run time 9.2 m Peak retention Rebaudioside M: 4.37 mtimes Rebaudioside I: 6.70 m Other glucosylated rebaudioside I product:4.8 m Second glucosylated rebaudioside I product: 6.7 m Column 40° C.temperature Injection volume 10 μL MS detection MRM 990/828 (for stevioltetraglycosides, e.g., rebaudioside A), 1152/828 (for steviolpentaglycosides, e.g., rebaudioside D), 1314/828 (steviolhexaglycosides, e.g., rebaudioside M), 828/666 (for stevioltriglycosides, e.g., stevioside), 666/504 (steviol diglycosides, e.g.,rubusoside) MS conditions MODE: MRM; CUR: 30; IS: 4750; CAD: high; TEM:550° C.; GS1: 50; GS2: 50; DP: 150; EP: 10; CXP: 14; DT: 50 ms for eachtransition. For the first three transitions CE: 85; for the last twotransitions CE: 60.

TABLE 44.2 β1,2GT Round 5 RebI Variants and RebM Levels SEQ ID NO: AminoAcid Differences Increased (nt/aa) (Relative to SEQ ID NO: 1002)RebM^(a) 2647/2648 F156R + 2649/2650 F156R/K161S + 2651/2652F156R/G199H/N200A + 2653/2654 F156R/K161S/N162G + 2663/2664F156R/K161S/N162G ++ 2665/2666 F156R/N162G/G199H ++ 2667/2668W21Y/L127H/P129A/K161S/N162G +++ 2669/2670 L127Q/P129A/K161S/G199H/N200A+++ 2671/2672 L127H/P129A/N162T + 2673/2674 F156R/K161S/N162G +2675/2676 L127Q/P129A/K161S/N162G/G199H +++ 2677/2678 F156R/K161S/N162G++ 2679/2680 W21Y/L127H/P129A/N162T/G199H/N200A +++ 2681/2682F156R/K161S/N162T ++ 2683/2684 W21Y/L127H/P129A/K1615 ++ ^(a)Levels ofincreased production were determined relative to the referencepolypeptide of SEQ ID NO: 1002, and defined as follows: “+” = productionat least 2.5-fold, but less than 3.13-fold; “++” = at least 3.13-fold,but less than 3.28-fold; and “+++” at least 3.28-fold increasedproduction, as compared to the reference polypeptide.

Example 45 Beta-1,2-ADP-Glycosyltransferase Variants of SEQ ID NO: 2600

In this Example, experiments for evolution and screening of GTpolypeptides derived from SEQ ID NO: 2600 for improved glucosylation ofsteviol glycosides using ADP-glucose are described. Directed evolutionof the GT encoded by SEQ ID NO: 2599 was carried out by constructinglibraries of variant genes. Libraries recombined mutations associatedwith the surface residues of the enzyme and beneficial mutationsassociated with improved activity in previous rounds, combinatoriallyincorporated diversity from homologs in publicly available databases, orsubjected certain structural features of the enzyme with saturationmutagenesis. These libraries were then plated, grown, and screened usingthe high-throughput (HTP) assay described below to provide a sixth round(“Round 6”) of engineered GT variant polypeptides withglucosyltransferase activity toward ADP-glucose and steviol glycosides.Twenty-four engineered variants were identified from the recombinedbeneficial mutations and homolog diversity (Table 45.1), and 21 wereidentified from saturation mutagenesis (Table 45.2).

HTP Assay for Glucose Transfer from ADP-Glucose to Rebaudioside A

Assays were performed on 96-well plates of cleared E. coli culturelysates expressing SEQ ID NO: 2600 variants. Lysis buffer volume was 400μL, and the lysate was diluted 40-fold. Assays were conducted with 10 μLdiluted lysate in 100 μL reaction volume with substrate loading of 1 mMrebaudioside A (>97% purity) and co-substrate loading of 1 mMADP-glucose (Sigma, >93% purity). The following reaction conditions wereused: 50 mM potassium phosphate buffer, pH 7, 3 mM MgCl₂, 45° C. in aThermotron® titre-plate shaker with 300 RPM shaking for 1 h. Thereactions were diluted 1:5 in water and then quenched by adding 25 μL ofthe diluted assay to 75 μL acetonitrile with 0.2% formic acid. Theresulting mixture was precipitated by centrifugation at 4° C. for 10 m.The supernatants were diluted 1:5 in water and analyzed by RapidFireSPE-MS/MS. The engineered variants that produced rebaudioside D fromrebaudioside A with ADP-glucose and that were identified from thecombinatorial libraries are listed in Table 45.1. The top 84 variantsfrom the saturation mutagenesis library were retested in triplicate asdescribed above at 50° C. The resulting engineered GT variantpolypeptides are listed in Table 45.2. Shake-flask scale cultures weregrown, lysed, and lyophilized to powder for variants listed in Table45.3.

TABLE 45.1 β1,2GT Round 6 Combinatorial Variants and RebD Levels SEQ IDNO: Amino Acid Differences Increased (nt/aa) (Relative to SEQ ID NO:2600) RebD^(a) 2685/2686 Al2S/R15K/I57K/E318D/L402I/A435V + 2687/2688I57K/T260V/E400Q/L402I/D404S + 2689/2690 I57K/L402I + 2691/2692A12S/P175S/E400Q/L402I/1406M ++ 2693/2694 I57K/E400Q/L402I + 2695/2696I57K/P175S/C220L/T260V/S264A/L402I ++ 2697/2698 A12S/I57K/P175S/K451N +2699/2700 H7E/Al2S/E400Q/A435V + 2701/2702 A12S/E318D/L402I/D404S/K451N++ 2703/2704 A12S/R15K/I57K/V71I/P175S/T260V/ +++ E400Q/L402I 2705/2706A12S/E318D/L402I/D404S/I406M/K451N +++ 2707/2708A12S/P175S/T260V/S264A/E318D/E400Q +++ 2709/2710A12S/R15K/E318D/E400Q/L402I/I406M + 2711/2712 T260V + 2713/2714I57K/P175S/D404G ++ 2715/2716 E400Q/L402I ++ 2717/2718H2-/H7E/A12S/R15K/P175S/T260V/E318D +++ 2719/2720R15K/P175S/E318D/E400Q/L402I ++ 2721/2722I57K/C220L/T260V/E400Q/L402I/I406M +++ 2723/2724A12S/R15K/I57K/C220L/T254K/T260V/ + E318D/L402I 2725/2726H7E/E318D/K451N + 2727/2728 L160V/F186M/R195P ++ 2729/2730 R195P +2731/2732 L152V/E192D/R195P + ^(a)Levels of increased production weredetermined relative to the reference polypeptide of SEQ ID NO: 2600, anddefined as follows: “+” = production at least 1.4-fold, but less than1.64-fold; “++” = at least 1.64-fold, but less than 1.84-fold; and “+++”at least 1.84-fold increased production, as compared to the referencepolypeptide.

TABLE 45.2 β1,2GT Round 6 Saturation Mutagenesis Variants and RebDLevels Amino Acid Differences Increased SEQ ID NO: (nt/aa) (Relative toSEQ ID NO: 2600) RebD^(a) 2733/2734 T240A +++ 2735/2736 I239F +2737/2738 R331S + 2739/2740 S148A ++ 2741/2742 T240P +++ 2743/2744I239E + 2745/2746 A327V +++ 2747/2748 V323L + 2749/2750 V135A ++2751/2752 F326M ++ 2753/2754 L152V +++ 2755/2756 F356G ++ 2757/2758F186V + 2759/2760 N32R ++ 2761/2762 D237T + 2763/2764 H325R + 2765/2766R331H + 2767/2768 P330A ++ 2769/2770 H325G + 2771/2772 I239Y + 2773/2774R331C + ^(a)Levels of increased production were determined relative tothe reference polypeptide of SEQ ID NO: 2600, and defined as follows:“+” = production at least 1.18-fold, but less than 1.4-fold; “++” = atleast 1.4-fold, but less than 1.6-fold; and “+++” at least 1.6-foldincreased production, as compared to the reference polypeptide.Shake Flask Powder Characterization Assay and Analysis for GlucosylTransfer from ADP-Glucose to Rebaudioside A

A shake flask powder loading dose response experiment was performed tocharacterize activity of the engineered round 6 variants on rebaudiosideA. The experiment was performed as described in Example 40, with levelsof 0.003-0.1 g/L SFP. The conversions of the round 6 variants onrebaudioside A at 0.025 g/L SFP loading are listed in Table 45.3. All 5of the variants listed in Table 45.3 had higher activities than SEQ IDNO: 2600. The variant with the mutations H2-, H7E, A12S, R15K, P175S,T260V, and E318D (SEQ ID NO: 2718) and its encoding polynucleotide (SEQID NO: 2717) were selected for further directed evolution for theglucosylation of rebaudioside A.

TABLE 45.3 β1,2GT Round 6 Shake Flask Powder Variants and RebD LevelsSEQ ID NO: Amino Acid Differences Increased (nt/aa) (Relative to SEQ IDNO: 2600) RebD^(a) 2687/2688 I57K/T260V/E400Q/L402I/D404S + 2695/2696157K/P175S/C220L/T260V/S264A/L402I + 2703/2704A12S/R15K/I57K/V71I/P175S/T260V/ + E400Q/L402I 2717/2718H2-/H7E/A12S/R15K/P175S/T260V/E318D ++ 2727/2728 L160V/F186M/R195P +^(a)Levels of increased production were determined relative to thereference polypeptide of SEQ ID NO: 2600, and defined as follows: “+” =production at least 1.1-fold, but less than 1.6-fold; and “++” = atleast 1.6-fold increased production, as compared to the referencepolypeptide.

Example 46 Beta-1,2-ADP-Glycosyltransferase Variants of SEQ ID NO: 2718

In this Example, experiments for evolution and screening of GTpolypeptides derived from SEQ ID NO: 2718 for improved glucosylation ofsteviol glycosides using ADP-glucose are described. Directed evolutionof the GT encoded by SEQ ID NO: 2717 was carried out by constructingcombinatorial libraries of variant genes in which beneficial mutationsassociated with improved activity or expression in previous rounds wererecombined. Another library subjected certain structural features of theenzyme to saturation mutagenesis. These libraries were then plated,grown, and screened using the high-throughput (HTP) assay describedbelow to provide a seventh round (“Round 7”) of engineered GT variantpolypeptides with glucosyltransferase activity toward ADP-glucose andsteviol glycosides. Twenty-five engineered variants were identified fromthe recombined beneficial mutations (Table 46.1) and 29 engineeredvariants were identified from saturation mutagenesis (Table 46.2).

HTP Coupled Assay for Glucose Transfer from Sucrose to ADP toRebaudioside A

Assays were performed on 96-well plates of cleared E. coli culturelysates expressing SEQ ID NO: 2717 variants. Lysis buffer volume was 400μL, and the lysate was diluted 10-fold. For screening the library inwhich beneficial mutations from previous rounds were recombined, assayswere conducted with 10 μL diluted lysate and 0.2 g/L SUS SFP SEQ ID NO:1392 in 100 μL reaction volume with substrate loading of 7.5 mMrebaudioside A and co-substrate loadings of 1 mM ADP (Sigma, >95%) and15 mM sucrose (Sigma). The following reaction conditions were used: 50mM potassium phosphate buffer, pH6.5, 50° C. in Thermotron® titre-plateshaker with 300 RPM shaking for 2h. The reactions were diluted 1:10 inwater and then quenched by adding 10 μL of the diluted assay to 90 μLacetonitrile with 0.2% formic acid. The resulting mixture wasprecipitated by centrifugation at 4° C. for 10 m. The supernatants werediluted 1:10 in water and analyzed by RapidFire SPE-MS/MS as describedin Table 31.1. For the remaining two round 7 libraries, screening wasperformed as described above, with the exceptions that 0.2 g/L SUS SFPSEQ ID NO: 1456 SFP and 30 mM sucrose were used. The engineered variantswith glucosyltransferase activity coupled with a SuS on rebaudioside Afrom the recombined beneficial mutations are listed in Table 46.1. Thetop variants from the saturation mutagenesis library were retested intriplicate, using the procedure described above with the exceptions that0.8 mM ADP was used, and the reactions were diluted 1:20 in water andthen quenched by adding 20 μL of the diluted assay to 80 μL acetonitrilewith 0.2% formic acid. The resulting engineered GT variant polypeptidesare listed in Table 46.2. Shake-flask scale cultures were grown, lysed,and lyophilized to powder for variants listed in Table 46.3.

TABLE 46.1 β1,2GT Round 7 Combinatorial Variants and RebD Levels SEQ IDNO: Amino Acid Differences Increased (nt/aa) (Relative to SEQ ID NO:2718) RebD^(a) 2775/2776 F185M/R194P/D236T/T239A/F325M/A326V ++2777/2778 K14R/M184A/F185M/R194P/D388E/ +++ E399Q/L401I 2779/2780K14R/R194P ++ 2781/2782 K14R/M184A/F185M/R194P/F234Y/F325M/ +++A326V/P329A 2783/2784 K14R/A326V/R330H +++ 2785/2786K14R/N31R/M184A/D274Q/V322L/A326V/ + P329A/R330H 2787/2788 K14R/F355G ++2789/2790 K14R/I56K/M184A/F185M/R194P/I238M/ +++ T239A/D274Q/P329A/E399Q2791/2792 K14R/F185M/D236T/I238M/T239A/D274Q/ ++V322L/A326V/P329Q/F355G/E399Q/L401I 2793/2794Kl4R/F185M/R194P/I238M/E399Q + 2795/2796 K14R/L23Q/D274Q/A326V/P329A +2797/2798 N31R/I56K/I315V/P329A/R330H + 2799/2800K14R/I56K/M184A/R194P/F234Y/I315V/ ++ A326V/P329Q/E399Q/L401I 2801/2802Kl4R/V322L/A326V/R330H + 2803/2804S147A/D236T/I238M/D243G/I315V/P329A/ + R330H/E399Q/L401I 2805/2806K14R/M184A/R194P/F355G/E399Q ++ 2807/2808K14R/I56K/G252D/D274Q/I315V/A326V/ + P329A/R330H/L401I 2809/2810L23Q/N31R/S147A/M184A/F185M/I238M/ ++G252D/F325M/P329A/R330H/D388E/L401I 2811/2812 K14R/F355G/E399Q +2813/2814 K14R/I56K/R194P/I238M/I315V/F325M/ ++ A326V/P329A/R330H/E399Q2815/2816 I238M + 2817/2818 E65D/I238T/N240S + 2819/2820 E65D +2821/2822 E65D/Q114E/H132R/I238M + 2823/2824 S223T/I412S + ^(a)Levels ofincreased production were determined relative to the referencepolypeptide of SEQ ID NO: 2718, and defined as follows: “+” = productionat least 1.05-fold, but less than 2.5-fold; “++” = at least 2.5-fold,but less than 3-fold; and “+++” at least 3-fold increased production, ascompared to the reference polypeptide.

TABLE 46.2 β1,2GT Round 7 Saturation Mutagenesis Variants and RebDLevels SEQ ID NO: Amino Acid Differences Increased (nt/aa) (Relative toSEQ ID NO: 2718) RebD^(a) 2825/2826 N391R ++ 2827/2828 K309E ++2829/2830 E65N +++ 2831/2832 M104L + 2833/2834 K422R + 2835/2836 I56T +2837/2838 K58R + 2839/2840 S11G +++ 2841/2842 L45V ++ 2843/2844 N138G ++2845/2846 N135L ++ 2847/2848 N138K +++ 2849/2850 H132Q ++ 2851/2852K309H + 2853/2854 E430L ++ 2855/2856 S11Q +++ 2857/2858 E165P +2859/2860 Y449F + 2861/2862 N286R +++ 2863/2864 Q114R + 2865/2866E256P + 2867/2868 E430V + 2869/2870 I55L + 2871/2872 H132S + 2873/2874I238G + 2875/2876 L113V + 2877/2878 E273R + 2879/2880 E65S ++ 2881/2882L45F ++ ^(a)Levels of increased production were determined relative tothe reference polypeptide of SEQ ID NO: 2718, and defined as follows:“+” = production at least that of the reference polypeptide, but lessthan 1.3-fold; “++” = at least 1.3-fold, but less than 1.7-fold; and“+++” at least 1.7-fold increased production, as compared to thereference polypeptide.Shake Flask Powder Characterization Assay and Analysis for GlucosylTransfer from ADP-Glucose to Rebaudioside A

A shake flask powder loading dose response experiment was performed tocharacterize activity of the engineered round 7 variants on rebaudiosideA. Levels of 0.002-0.2 g/L shake flask powder (SFP) were added to a 100μL total reaction volume containing 50 mM potassium phosphate buffer, pH6.5, 2 mM rebaudioside A, and 2 mM ADP-glucose. The reaction wasperformed at 50′C in a Thermotron® titre-plate shaker at 300 RPM for 1h.The reaction was diluted 1:5 in water and then quenched by adding 25 μLof the diluted assay to 75 μL acetonitrile with 0.2% formic acid andprecipitated by centrifugation at 4° C. for 10 m. The supernatant wasdiluted 1:10 in water and analyzed for steviol glycosides by RapidFireSPE-MS/MS. The conversions of the round 7 variants on rebaudioside A at0.025 g/L SFP loading are shown in Table 46.3. All 7 of the variantslisted in Table 46.3 had higher activities than SEQ ID NO: 2718. Thevariant with the mutations K14R, 156K, R194P, 1I238M, 1I315V, F325M,A326V, P329A, R330H, and E399Q (SEQ ID NO: 2814) and its encodingpolynucleotide (SEQ ID NO: 2813) were selected for further directedevolution for the glucosylation of rebaudioside A.

TABLE 46.3 β1,2GT Round 7 Shake Flask Powder Variants and RebD LevelsSEQ ID NO: Amino Acid Differences Increased (nt/aa) (Relative to SEQ IDNO: 2718) RebD^(a) 2777/2778 K14R/M184A/F185M/R194P/D388E/E399Q/ ++L401I 2793/2794 K14R/F185M/R194P/I238M/E399Q ++ 2799/2800K14R/I56K/M184A/R194P/F234Y/I315V/ + A326V/P329Q/E399Q/L401I 2805/2806K14R/M184A/R194P/F355G/E399Q + 2809/2810L23Q/N31R/S147A/M184A/F185M/I238M/ +++G252D/F325M/P329A/R330H/D388E/L401I 2811/2812 K14R/F355G/E399Q ++2813/2814 K14R/I56K/R194P/I238M/I315V/F325M/ +++ A326V/P329A/R330H/E399Q^(a)Levels of increased production were determined relative to thereference polypeptide of SEQ ID NO: 2718, and defined as follows: “+” =production at least 1.6-fold, but less than 2.3-fold; “++” = at least2.3-fold, but less than 2.5-fold; and “+++” at least 2.5-foldproduction, as compared to the reference polypeptide.

Example 47 Beta-1,2-ADP-Glycosyltransferase Variants of SEQ ID NO: 2814

In this Example, experiments for evolution and screening of GTpolypeptides derived from SEQ ID NO: 2814 for improved glucosylation ofsteviol glycosides using ADP-glucose are described. Directed evolutionof the GT encoded by SEQ ID NO: 2813 was carried out by constructingcombinatorial libraries of variant genes in which beneficial mutationsassociated with improved activity in previous rounds were recombined andin which certain structural features of the enzyme were subjected withsaturation mutagenesis. These libraries were then plated, grown, andscreened using the high-throughput (HTP) assay described below toprovide an eighth round (“Round 8”) of engineered GT variantpolypeptides with glucosyltransferase activity toward ADP-glucose andsteviol glycosides. Sixteen engineered variants were identified from therecombined beneficial mutations (Table 47.1), and 18 were identifiedfrom saturation mutagenesis (Table 47.2).

HTP Coupled Assay for Glucose Transfer from Sucrose to ADP toRebaudioside A

Assays were performed on 96-well plates of cleared E. coli culturelysates expressing SEQ ID NO: 2813 variants. Lysis buffer volume was 400μL, and the lysate was diluted 20-fold. For screening the library inwhich beneficial mutations from previous rounds were recombined, assayswere conducted with 10 μL diluted lysate and 0.2 g/L SUS SFP SEQ ID NO:1456 in 100 μL reaction volume with substrate loading of 7.5 mMrebaudioside A and co-substrate loadings of 0.4 mM ADP (Sigma, >95%) and30 mM sucrose (Sigma). The following reaction conditions were used: 50mM potassium phosphate buffer, pH 6.5, 50° C. in a Thermotron®titre-plate shaker with 300 RPM shaking for 1 h. The reactions werediluted 1:20 in water and then quenched by adding 20 μL of the dilutedassay to 80 μL acetonitrile with 0.2% formic acid. The resulting mixturewas precipitated by centrifugation at 4° C. for 10 m. The supernatantswere diluted 1:10 in water and analyzed by RapidFire SPE-MS/MS. Theresulting engineered variants with glucosyltransferase activity coupledwith a SuS on rebaudioside A are listed in Table 47.1. For the round 8saturation mutagenesis library, screening was performed as describedabove with the exceptions that 0.2 g/L SUS SFP SEQ ID NO: 1582, 0.2 mMADP, and the following reaction conditions were used: 50 mM potassiumphosphate buffer, pH 6, 55° C. in a Thermotron® titre-plate shaker with300 RPM shaking for 2 h. The top 56 variants from this library wereretested in triplicate using the same assay conditions with theexception that 0.15 g/L SUS SFP SEQ ID NO: 1582 was used. The resultingengineered GT variant polypeptides are listed in Table 47.2. Shake-flaskscale cultures were grown, lysed, and lyophilized to powder for variantslisted in Table 47.3.

TABLE 47.1 β1,2GT Round 8 Combinatorial Variants and RebD Levels SEQ IDNO: Amino Acid Differences Increased (nt/aa) (Relative to SEQ ID NO:2814) RebD^(a) 2883/2884 N31R/D388E ++ 2885/2886 M238Y/H324G/A329Q/F355G++ 2887/2888 N31R/V134A/F185M/G252D/D274Q/H324G/ +++ D388E 2889/2890L23Q/N31R/F185M/H324G/L401I + 2891/2892 N31R/V134A/G252D/H324G +2893/2894 N31R/V134A/M238E/G252D/V322L/H324G/ ++ D388E 2895/2896H324G/A329Q/F355G/L401I + 2897/2898 V134A/M184A/F185M/F234Y/D236T/T239A/+++ D274Q/H324G/D388E 2899/2900 N31R/V322L ++ 2901/2902 D236T +2903/2904 F234Y/D236T/M238E/V322L/H324G/F355G +++ 2905/2906V322L/H324G + 2907/2908 M184A/F185M/V322L + 2909/2910 N31R/D236T +2911/2912 L23Q/N31R/F185M/F355G + 2913/2914N31R/M184A/F185M/M238Y/T239A/V322L + ^(a)Levels of increased productionwere determined relative to the reference polypeptide of SEQ ID NO:2814, and defined as follows: “+” = production at least 1.3-fold that ofthe reference polypeptide, but less than 1.4-fold; “++” = at least1.4-fold but less than 1.7-fold; and “+++” = greater than 1.7-foldincreased production, as compared to the reference polypeptide.

TABLE 47.2 β1,2GT Round 8 Saturation Mutagenesis Variants and RebDLevels SEQ ID NO: Amino Acid Differences Increased (nt/aa) (Relative toSEQ ID NO: 2814) RebD^(a) 2915/2916 I427R + 2917/2918 S426R +++2919/2920 V164H ++ 2921/2922 I440R ++ 2923/2924 M325L +++ 2925/2926V176R +++ 2927/2928 V164R + 2929/2930 S446R ++ 2931/2932 T400V ++2933/2934 I122L + 2935/2936 S426A +++ 2937/2938 V164M + 2939/2940 V176K++ 2941/2942 V176N + 2943/2944 V176L + 2945/2946 E177A + 2947/2948G316R + 2949/2950 K425R + ^(a)Levels of increased production weredetermined relative to the reference polypeptide of SEQ ID NO: 2814, anddefined as follows: “+” = production at least that of the referencepolypeptide, but less than 1.25-fold; “++” = at least 1.25-fold, butless than 1.35-fold; and “+++” = at least 1.35-fold increasedproduction, as compared to the reference polypeptide.Shake Flask Powder Characterization Assay and Analysis for GlucosylTransfer from Sucrose to ADP to Rebaudioside A

A shake flask powder loading dose response experiment was performed tocharacterize activity of the engineered round 8 variants on rebaudiosideA. Levels of 0.002-0.2 g/L shake flask powder (SFP) were added to a 100μL total reaction volume containing 50 mM potassium phosphate buffer, pH6.5, 8 mM rebaudioside A, 0.4 mM ADP, 30 mM sucrose, and 0.2 g/L SUS SFPSEQ ID NO: 1456. The reaction was performed at 50° C. in a Thermotron®titre-plate shaker at 300 RPM for 1 h. The reaction was diluted 1:20 inwater and then quenched by adding 20 μL of the diluted assay to 80 μLacetonitrile with 0.2% formic acid and precipitated by centrifugation at4° C. for 10 m. The supernatant was diluted 1:10 in water and analyzedfor steviol glycosides by RapidFire SPE-MS/MS. The conversion of theround 8 variants on rebaudioside A at 0.1 g/L SFP loading are shown inTable 47.3. All 8 of the variants listed in Table 47.3 had higheractivities than SEQ ID NO: 2814. The variant with the mutations N31R andD388E (SEQ ID NO:2884) and its encoding polynucleotide (SEQ ID NO: 2883)were selected for further directed evolution for the glucosylation ofrebaudioside A.

TABLE 47.3 β1,2GT Round 8 Shake Flask Variants and RebD Levels SEQ IDNO: Amino Acid Differences Increased NO: (nt/aa) (Relative to SEQ ID NO:2814) RebD^(a) 2883/2884 N31R/D388E +++ 2887/2888N31R/V134A/F185M/G252D/D274Q/H324G/ + D388E 2893/2894N31R/V134A/M238E/G252D/V322L/H324G/ + D388E 2897/2898V134A/M184A/F185M/F234Y/D236T/T239A/ + D274Q/H324G/D388E 2899/2900N31R/V322L ++ 2903/2904 F234Y/D236T/M238E/V322L/H324G/F355G ++ 2909/2910N31R/D236T + 2913/2914 N31R/M184A/F185M/M238Y/T239A/V322L +++ ^(a)Levelsof increased production were determined relative to the referencepolypeptide of SEQ ID NO: 2814, and defined as follows: “+” = productionat least 1.2-fold, less than 1.7-fold; “++” = at least 1.7-fold, butless than 1.9-fold; and “+++” = at least 1.9-fold increased production,as compared to the reference polypeptide.

Example 48 Beta-1,2-ADP-Glycosyltransferase Variants of SEQ ID NO: 2884

In this Example, experiments for evolution and screening of GTpolypeptides derived from SEQ ID NO: 2884 for improved glucosylation ofsteviol glycosides using ADP-glucose are described. Directed evolutionof the GT encoded by SEQ ID NO: 2883 was carried out by constructingcombinatorial libraries of variant genes in which beneficial mutationsassociated with improved activity in previous rounds were recombined.These libraries were then plated, grown, and screened using thehigh-throughput (HTP) assay described below to provide a ninth round(“Round 9”) of 33 engineered GT variant polypeptides withglucosyltransferase activity toward ADP-glucose and steviol glycosides.

HTP Coupled Assay for Glucose Transfer from Sucrose to ADP toRebaudioside A

Assays were performed on 96-well plates of cleared E. coli culturelysates expressing SEQ ID NO: 2883 variants. Lysis buffer volume was 400μL, and the lysate was diluted 20-fold. Assays were conducted with 10 μLdiluted lysate and 0.15 g/L SUS SFP SEQ ID NO: 1582 in 100 μL reactionvolume with substrate loading of 7.5 mM rebaudioside A and co-substrateloadings of 0.2 mM ADP (Sigma, >95%) and 24 mM sucrose (Sigma). Thefollowing reaction conditions were used: 50 mM potassium phosphatebuffer, pH 6, 55° C. in a Thermotron® titre-plate shaker with 300 RPMshaking for 2 h. The reactions were diluted, quenched, and analyzed asdescribed above. The resulting engineered variants withglucosyltransferase activity coupled with a SuS on rebaudioside A arelisted in Table 48.1. Shake-flask scale cultures were grown, lysed, andlyophilized to powder for variants listed in Table 48.2.

TABLE 48.1 β1,2GT Round 9 Combinatorial Variants and RebD Levels SEQ IDNO: Amino Acid Differences Increased (nt/aa) (Relative to SEQ ID NO:2884) RebD^(a) 2951/2952 S11Q/K58R/E65N/M104L/P107G/ + N138G/N286R2953/2954 S11G/K58R/H132Q/E165P/N286R/N391R/ + K422S/E430L 2955/2956N135L/N138G/E165P/K309H/E430L + 2957/2958S11Q/E65N/M104L/P107G/N138G/E165P/ ++ N286R/N391R/E430L 2959/2960P107G/Q114R/H132Q/N138G + 2961/2962 S11Q/K58R + 2963/2964K58R/E65N/E165P/K309H/E430L ++ 2965/2966 S11Q + 2967/2968S11Q/K58R/E65N/N135L/N138G ++ 2969/2970 K58R/M104L/Q114R/E165P/N391R +2971/2972 S11Q/N138K/E165P/S223T/K309H + 2973/2974S11Q/E65N/P107G/N135L/E165P/N391R/ + E430L 2975/2976 S11Q/P107G/N138G +2977/2978 S11Q/E65N/N135L/N138G + 2979/2980S11G/E65N/H132Q/N391R/E430L + 2981/2982 S11Q/M104L/H132Q/N138K/N391R +2983/2984 S11Q/Q114R/S223T/G252D/N286R/N391R ++ 2985/2986S11G/M104L/H132Q/N138G/K309H/N391R ++ 2987/2988S11G/K58R/M104L/P107G/Q114R/N138K/ +++ S223T/N391R 2989/2990S11G/L45V/K58R/H132Q/N138G/N286R ++ 2991/2992S11G/P107G/Q114R/S223T/K309H/E430L +++ 2993/2994S11Q/H132Q/N135L/N138K/S223T/N286R +++ 2995/2996 S223T/K309E ++2997/2998 S11Q/H132Q/N138G/S223T/N286R/N391R +++ 2999/3000K58R/E65N/N138K ++ 3001/3002 P107G/E430L + 3003/3004S11Q/K58R/N138G/K309E +++ 3005/3006 S11G/K58R/Q114R/N286R/K309H/N391R +3007/3008 K58R/E65N/N138G/E165P ++ 3009/3010S11Q/E65N/H132Q/N135L/G252D + 3011/3012 S11Q/Q114R/N135L/N138G/S223T ++3013/3014 S11Q/K58R/E65N/P107G/N135L/S223T/ + N286R/N391R/E430L3015/3016 S11Q/E65N/H132Q/N135L/N138G/S223T/ +++ N391R ^(a)Levels ofincreased production were determined relative to the referencepolypeptide of SEQ ID NO: 2884, and defined as follows: “+” = productionat least 1.5-fold, but less than 2.1-fold; “++” = at least 2.1-fold, butless than 2.5-fold; and “+++” = at least 2.5-fold increased production,relative to reference polypeptide.Shake Flask Powder Characterization Assay and Analysis for GlucosylTransfer from Sucrose to ADP to Rebaudioside A

A shake flask powder loading dose response experiment was performed tocharacterize activity of the engineered round 9 variants on rebaudiosideA. Levels of 0.002-0.2 g/L shake flask powder (SFP) were added to a 100μL total reaction volume containing 50 mM potassium phosphate buffer,pH6, 8 mM rebaudioside A, 0.4 mM ADP, 24 mM sucrose, and 0.15 g/L SUSSFP SEQ ID NO: 1582. The reaction was performed at 55° C. in aThermotron® titre-plate shaker at 300 RPM for 1 h. The reactions werediluted, quenched, and analyzed as described above. The conversion ofthe round 9 variants on rebaudioside A at 0.05 g/L SFP loading are shownin Table 48.2. All 6 of the variants listed in Table 48.2 had higheractivities than SEQ ID NO: 2884. The variant with the mutation S11Q,E65N, H132Q, N135L, N138G, S223T, and N391R (SEQ ID NO:3016) and itsencoding polynucleotide (SEQ ID NO: 3015) were most improved and wereselected for further directed evolution for the glucosylation ofrebaudioside A.

TABLE 48.2 β1,2GT Round 9 Shake Flask Powder Variants and RebD LevelsSEQ ID NO: Amino Acid Differences Increased (nt/aa) (Relative to SEQ IDNO: 2884) RebD^(a) 3015/3016 S11Q/E65N/H132Q/N135L/N138G/S223T/ +++N391R 2983/2984 S11Q/Q114R/S223T/G252D/N286R/N391R ++ 2997/2998S11Q/H132Q/N138G/S223T/N286R/N391R + 2987/2988S11G/K58R/M104L/P107G/Q114R/N138K/ ++ S223T/N391R 3011/3012S11Q/Q114R/N135L/N138G/S223T + 2975/2976 S11Q/P107G/N138G + ^(a)Levelsof increased production were determined relative to the referencepolypeptide of SEQ ID NO: 2884, and defined as follows: “+” = productionat least 1.07-fold, but less than 1.2-fold; “++” = at least 1.2-fold,but less than 1.85-fold; and “+++” = at least 1.85-fold increasedproduction, as compared to the reference polypeptide.

Example 49 Beta-1,2-ADP-Glycosyltransferase Variants of SEQ ID NO: 3016

In this Example, experiments for evolution and screening of GTpolypeptides derived from SEQ ID NO: 3016 for improved glucosylation ofsteviol glycosides using ADP-glucose are described. Directed evolutionof the GT encoded by SEQ ID NO: 3015 was carried out by constructinglibraries of variant genes. Libraries recombined mutations associatedwith improved activity in previous rounds and subjected certainstructural features of the enzyme with saturation mutagenesis. Theselibraries were then plated, grown, and screened using thehigh-throughput (HTP) assay described below to provide a tenth round(“Round 10”) of engineered GT variant polypeptides withglucosyltransferase activity toward ADP-glucose and steviol glycosides.Forty engineered variants were identified from the recombined beneficialmutations (Table 49.1), and 40 were identified from saturationmutagenesis (Table 49.2).

HTP Coupled Assay for Glucose Transfer from Sucrose to ADP toRebaudioside A

Assays were performed on 96-well plates of cleared E. coli culturelysates expressing SEQ ID NO: 3015 variants. Lysis buffer volume was 400μL, and the lysate was diluted 20-fold. For screening the library inwhich beneficial mutations from previous rounds were recombined, assayswere conducted with 10 μL diluted lysate and 0.15 g/L SUS SFP SEQ ID NO:1764 in 100 μL reaction volume with substrate loading of 8 mMrebaudioside A and co-substrate loadings of 0.2 mM ADP (Sigma, >95%) and24 mM sucrose. The following reaction conditions were used: 50 mMpotassium phosphate buffer, pH 6, 55° C. in a Thermotron® titre-plateshaker with 300 RPM shaking for 1 h. The reactions were diluted 1:20 inwater and then quenched by adding 20 μL of the diluted assay to 80 μLacetonitrile with 0.2% formic acid. The resulting mixture wasprecipitated by centrifugation at 4° C. for 10 m. The supernatants werediluted 1:10 in water and analyzed by RapidFire SPE-MS/MS. The resultingengineered variants with glucosyltransferase activity coupled with a SuSon rebaudioside A are listed in Table 49.1. For the round 10 saturationmutagenesis library, screening was performed as described above with theexceptions that the lysate was diluted 10-fold, and the followingreaction conditions were used: 60° C. in a Thermotron® titre-plateshaker with 300 RPM shaking for 2 h. The resulting engineered GT variantpolypeptides are listed in Table 49.2. Shake-flask scale cultures weregrown, lysed, and lyophilized to powder for variants listed in Table49.3.

TABLE 49.1 β1,2GT Round 10 Combinatorial Variants and RebD Levels SEQ IDNO: Amino Acid Differences Increased (nt/aa) (Relative to SEQ ID NO:3016) RebD^(a) 3017/3018 K58R/I122L/I440R + 3019/3020K58R/I122L/V164H/D236T/S446R + 3021/3022K58R/P107G/I122L/V322L/T400V/K425R/ ++ I427R/I440R/S446R 3023/3024K58R/P107G/D236T/T400V + 3025/3026 I122L/V164H/V176R/H324G/T400V +++3027/3028 I122L/V176R/D236T/T400V ++ 3029/3030P107G/V164H/D236T/T400V/S446R ++ 3031/3032 V164H ++ 3033/3034K58R/V176R/D236T +++ 3035/3036 V176R/T400V +++ 3037/3038K58R/P107G/I122L/D236T/H324G/T400V/ ++ K425R/S446R 3039/3040V164H/V322L/H324G/M325L/T400V/ + K425R/I440R/S446R 3041/3042 I122L/T400V+++ 3043/3044 K58R/V164H/V176R +++ 3045/3046 P107G/I440R + 3047/3048P107G/I122L/D236T/K425R/S426A/S446R +++ 3049/3050P107G/V176R/V322L/M325L/I440R/S446R ++ 3051/3052 I122L/V164H/T400V/I440R+++ 3053/3054 K58R/P107G/I122L/V176R/D236T/H324G/ +M325L/T400V/S426R/I427R 3055/3056 I122L/V164H/T400V/I440R/S446R +3057/3058 K58R/P107G/T400V + 3059/3060H324G/T400V/K425R/S426R/I440R/S446R ++ 3061/3062 I122L/I440R/S446R +++3063/3064 V164H/S271G/K425R/S426R ++ 3065/3066 V164H/T400V ++ 3067/3068P107G/D236T ++ 3069/3070 K58R/P107G/V164H/T400V ++ 3071/3072K58R/I122L/M325L + 3073/3074 K58R/I122L/V322L/M325L ++ 3075/3076K58R/I122L/V176R/D236T/T400V/S446R + 3077/3078K58R/V164H/H324G/K425R/I427R + 3079/3080P107G/V176R/T400V/K425R/I427R/I440R + 3083/3084 K58R/D236T + 3085/3086I122L/K425R/S426R/S446R + 3087/3088 I122L/V176R/H324G/T400V/I440R +3089/3090 K58R/I122L/V176R/V322L/H324G/S426A/ + I427R 3093/3094I139V/G252D + 3095/3096 G8S/K448A + 3097/3098 I139V + 3099/3100 P189R +^(a)Levels of increased production were determined relative to thereference polypeptide of SEQ ID NO: 3016, and defined as follows: “+” =production at least 1.2-fold, but less than 1.43-fold; “++” = at least1.43-fold, but less than 1.67-fold; and “+++” = at least 1.67-foldincreased production, as compared to the reference polypeptide.

TABLE 49.2 β1,2GT Round 10 Saturation Mutagenesis Variants and RebDLevels SEQ ID NO: Amino Acid Differences Increased (nt/aa) (Relative toSEQ ID NO: 3016) RebD^(a) 3101/3102 N240E +++ 3103/3104 A242Y +3105/3106 S9C + 3107/3108 E210V +++ 3109/3110 E408P ++ 3111/3112 G200T++ 3113/3114 N115R + 3115/3116 N172R ++ 3117/3118 A242I ++ 3119/3120L116V + 3121/3122 G415R +++ 3123/3124 K245M + 3125/3126 G200A +++3127/3128 E411Q + 3129/3130 A213G + 3131/3132 G415K ++ 3133/3134 T409L+++ 3135/3136 N423R +++ 3137/3138 N172S + 3139/3140 N65S + 3141/3142K106A ++ 3143/3144 Q178K + 3145/3146 N423A ++ 3147/3148 N447R +3149/3150 D255P +++ 3151/3152 N240C ++ 3153/3154 S9M ++ 3155/3156H324R/N423R +++ 3157/3158 N447L + 3159/3160 K416R + 3161/3162 V385R +3163/3164 N115D + 3165/3166 N240L ++ 3167/3168 E210L ++ 3169/3170 G415A++ 3171/3172 G200V + 3173/3174 N240P + 3175/3176 N240V + 3177/3178I412R + 3179/3180 G415H + ^(a)Levels of increased production weredetermined relative to the reference polypeptide of SEQ ID NO: 3016, anddefined as follows: “+” = production at least 1.1-fold that of thereference polypeptide, but less than 1.38-fold; “++” = at least1.38-fold but less than 1.76-fold; and “+++” = at least 1.76-foldproduction, relative to reference polypeptide.Shake Flask Powder Characterization Assay and Analysis for GlucosylTransfer from Sucrose to ADP to Rebaudioside A

A shake flask powder loading dose response experiment was performed tocharacterize activity of the engineered round 10 variants onrebaudioside A. Levels of 0.002-0.2 g/L shake flask powder (SFP) wereadded to a 100 μL total reaction volume containing 50 mM potassiumphosphate buffer, pH 6, 8 mM rebaudioside A, 0.4 mM ADP, 24 mM sucrose,and 0.15 g/L SUS SFP SEQ ID NO: 1764. The reaction was performed at 60°C. in a Thermotron® titre-plate shaker at 300 RPM for 1 h. The reactionwas diluted 1:20 in water and then quenched by adding 20 μL of thediluted assay to 80 μL acetonitrile with 0.2% formic acid andprecipitated by centrifugation at 4° C. for 10 m. The supernatant wasdiluted 1:10 in water and analyzed for steviol glycosides by RapidFireSPE-MS/MS. The activities of the round 10 variants on rebaudioside A at0.25 g/L SFP loading are shown in Table 49.3. All 5 of the variantslisted in Table 49.3 had higher activities than SEQ ID NO: 3016. Thevariant with the mutations K58R, I122L, V176R, T400V, K425R, S426A,I427R, and S446R (SEQ ID NO: 3082) and its encoding polynucleotide (SEQID NO: 3081) were most improved and were selected for further directedevolution for the glucosylation of rebaudioside A.

TABLE 49.3 β1,2GT Round 10 Shake Flask Variants and RebD Levels SEQ IDNO: Amino Acid Differences Increased (nt/aa) (Relative to SEQ ID NO:3016) RebD^(a) 3061/3062 I122L/I440R/S446R + 3081/3082K58R/I122L/V176R/T400V/K425R/ ++ S426A/I427R/S446R 3089/3090K58R/I122L/V176R/V322L/H324G/ + S426A/I427R 3091/3092 K448A + 3099/3100P189R + ^(a)Levels of increased production were determined relative tothe reference polypeptide of SEQ ID NO: 3016, and defined as follows:“+” = production at least that of the reference polypeptide, but lessthan 1.3-fold; and “++” = at least 1.3-fold increased production, ascompared to the reference polypeptide.

Example 50 Beta-1,2-ADP-Glycosyltransferase Variants of SEQ ID NO: 3082

In this Example, experiments for evolution and screening of GTpolypeptides derived from SEQ ID NO: 3082 for improved glucosylation ofsteviol glycosides using ADP-glucose are described. Directed evolutionof the GT encoded by SEQ ID NO: 3081 was carried out by constructingcombinatorial libraries of variant genes. Libraries recombined mutationsassociated with improved activity in previous rounds and subjectedcertain structural features of the enzyme with saturation mutagenesis.These libraries were then plated, grown, and screened using the HTPassay described below, to provide an eleventh round (“Round 11”) ofengineered GT variant polypeptides with glucosyltransferase activitytoward ADP-glucose and steviol glycosides. Fifty engineered variantswere identified from the recombined beneficial mutations (Table 50.1),and 53 were identified from saturation mutagenesis (Table 50.2).

HTP Coupled Assay for Glucose Transfer from Sucrose to ADP toRebaudioside A

Assays were performed on 96-well plates of cleared E. coli culturelysates expressing SEQ ID NO: 3081 variants. Lysis buffer volume was 400μL, and the lysate was diluted 20-fold. For screening the library inwhich beneficial mutations from previous rounds were recombined, assayswere conducted with 10 μL diluted lysate and 0.15 g/L SUS SFP SEQ ID NO:1764 in 100 μL reaction volume with substrate loading of 8 mMrebaudioside A and co-substrate loadings of 0.2 mM ADP (Sigma, >95%) and24 mM sucrose. The following reaction conditions were used: 50 mMpotassium phosphate buffer, pH 6, 60° C. in a Thermotron® titre-plateshaker with 300 RPM shaking for 2 h. The reactions were diluted,quenched, and analyzed as described above. The resulting engineeredvariants with glucosyltransferase activity coupled with a SuS onrebaudioside A are listed in Table 50.1. For the round 11 saturationmutagenesis library, screening was performed as described above with theexception that a substrate loading of 10 mM rebaudioside A was used. Theresulting engineered GT variant polypeptides are listed in Table 50.2.Shake-flask scale cultures were grown, lysed, and lyophilized to powderfor several variants.

TABLE 50.1 β1,2GT Round 11 Combinatorial Variants and RebD Levels SEQ IDNO: Amino Acid Differences Increased (nt/aa) (Relative to SEQ ID NO:3082) RebD^(a) 3181/3182 V164H/G200A/D236T +++ 3183/3184G8S/I139V/G200A/D236T/N240E/D255P/ +++ N423R 3185/3186P107G/D236T/N240E/M325L/I440R + 3187/3188 V164H/V322L/M325L/K416R/N423R+++ 3189/3190 G8S/P107G/V164H/D255P ++ 3191/3192P107G/I139V/G200A/N240E/V322L/H324G/ + M325L/K448A 3193/3194 G8S ++3195/3196 V164H/P189R/D236T/N240E ++ 3197/3198 V164H/D236T/N423R +3199/3200 V164H/D236T/N240E/I440R + 3201/3202G8S/P107G/I139V/V164H/G415A/K416R/ + I440R/K448A 3203/3204G8S/V164H/N240E/N423R +++ 3205/3206 P107G/N423R ++ 3207/3208T12S/V164H/I440R ++ 3209/3210 V164H/D236T + 3211/3212G8S/I139V/V164H/G200A/D236T/N240E/ ++ V322L/I440R/K448A 3213/3214V164H/P189R/N240E/G252D/G415A/N423R + 3215/3216V164H/G200A/D236T/D255P/V322L/H324G/ +++ N423R/I440R 3217/3218 I139V +3219/3220 G8S/P107G/I139V/D255P/V322L/M325L/ +++ G415A/K416R/I440R/K448A3221/3222 V164H/P189R + 3223/3224 G8S/V164H/P189R/G200A/V322L/H324G/ +M325L/K416R/K448A 3225/3226 G8S/D236T/N240E/G252D/K448A + 3227/3228G8S/I139V/D255P/G415A/K416R ++ 3229/3230 I139V/V164H/D236T/N240E +3231/3232 P107G/N240E/G252D/N423R/K448A ++ 3233/3234G8S/I139V/V164H/D236T/K416R/N423R/ + I440R 3235/3236N240E/G252D/D255P/G415A/K448A +++ 3237/3238N240E/G252D/D255P/V322L/G415A/K416R ++ 3239/3240 G8S/I139V + 3241/3242V164H/G200A/D236T/N240E/H324G/K416R/ + I440R 3243/3244G8S/G252D/D255P/V322L/M325L/K448A + 3245/3246G200A/D236T/V322L/M325L/K416R + 3247/3248 G200A/V322L/M325L/G415A/K448A++ 3249/3250 G415A/K416R/K448A ++ 3251/3252 G8S/V164H/K448A + 3253/3254I139V/D255P/M325L/G415A/I440R ++ 3255/3256 P107G/I139V + 3257/3258G8S/V164H/G252D/D255P/K448A + 3259/3260 G8S/V164H + 3261/3262D236T/G415A/K416R + 3263/3264 G8S/P107G/V164H/N423R/I440R + 3265/3266V164H/K416R/K448A ++ 3267/3268 V164H/V322L/M325L ++ 3269/3270G8S/I139V/P189R/N240E/M325L/K416R + 3271/3272 G8S/N240E/N423R +++3273/3274 D255P/N423R +++ 3275/3276 M325L + 3277/3278 N240E + 3279/3280H2- +++ ^(a)Levels of increased production were determined relative tothe reference polypeptide of SEQ ID NO: 3082, and defined as follows:“+” = production at least 1.25-fold, but less than 1.44-fold; “++” = atleast 1.44-fold, but less than 1.62-fold; and “+++” = at least 1.62-foldincreased production, as compared to the reference polypeptide.

TABLE 50.2 β1,2GT Round 11 Saturation Mutagenesis Variants and RebDLevels SEQ ID NO: Amino Acid Differences Increased (nt/aa) (Relative toSEQ ID NO: 3082) RebD^(a) 3385/3386 H73T + 3387/3388 N286L + 3389/3390F355A ++ 3391/3392 P75H + 3393/3394 D389F ++ 3395/3396 E299L ++3397/3398 H2T + 3399/3400 L113I +++ 3401/3402 H73A +++ 3403/3404 K309R++ 3405/3406 P237M + 3407/3408 R446H + 3409/3410 H2N ++ 3411/3412 P237V++ 3413/3414 T406N +++ 3415/3416 H73P + 3417/3418 E299R + 3419/3420E256I + 3421/3422 D323P + 3423/3424 P189S + 3425/3426 T406F + 3427/3428H2S + 3429/3430 T406Q ++ 3431/3432 E299A + 3433/3434 K34R ++ 3435/3436N286S +++ 3437/3438 D305G + 3439/3440 E313D + 3441/3442 E256S ++3443/3444 H73V + 3445/3446 E256T + 3447/3448 E186G +++ 3449/3450 K422R++ 3451/3452 G8S + 3453/3454 E186I + 3455/3456 T406G + 3457/3458 R446P++ 3459/3460 T239A ++ 3461/3462 K422S + 3463/3464 E299V + 3465/3466P237L ++ 3467/3468 L312V ++ 3469/3470 Q114V +++ 3471/3472 H3I +3473/3474 E221K + 3475/3476 L72Y ++ 3477/3478 L312S +++ 3479/3480 Q235M+++ 3481/3482 E256L +++ 3483/3484 D389G + 3485/3486 K422C + 3487/3488L312T +++ 3489/3490 E438T +++ ^(a)Levels of increased production weredetermined relative to the reference polypeptide of SEQ ID NO: 3082, anddefined as follows: “+” = production at least 1.15-fold, but less than1.2-fold; “++” = at least 1.2-fold, but less than 1.27-fold; and “+++” =at least 1.27-fold increased production, as compared to the referencepolypeptide.Shake Flask Powder Characterization Assay and Analysis for GlucosylTransfer from Sucrose to ADP to Rebaudioside A

A shake flask powder loading dose response experiment was performed tocharacterize activity of the engineered round 11 variants onrebaudioside A. Levels of 0.006-0.2 g/L shake flask powder (SFP) wereadded to a 100 μL total reaction volume containing 50 mM potassiumphosphate buffer, pH 6, 10 mM rebaudioside A, 0.2 mM ADP, 24 mM sucrose,and 0.15 g/L SUS SFP SEQ ID NO: 1764. The reaction was performed at 60°C. in a Thermotron® titre-plate shaker at 300 RPM for 2 h. The reactionswere diluted, quenched, and analyzed as described above. The round 11variant that was most improved on rebaudioside A at 0.025 g/L SFPloading was SEQ ID NO: 3244, which had mutations G8S, G252D, D255P,V322L, M325L, and K448A relative to SEQ ID NO: 1764. This variant, SEQID NO: 3244, was used for further directed evolution.

HTP Coupled Assay for Glucose Transfer from Sucrose to ADP toRebaudioside I

Directed evolution of the GT encoded by SEQ ID NO: 3081 was carried outby constructing libraries of variant genes in which mutations associatedwith improved glycosyltransferase activity toward rebaudioside I inprevious rounds were recombined. These libraries were then plated,grown, and screened using the high-throughput (HTP) assay describedbelow to provide a round (“Round 11.04”) of engineered GT variantpolypeptides with glucosyltransferase activity toward ADP-glucose andrebaudioside I. Cells were lysed with 400 μL lysis buffer. Assays wereperformed on 96-well plates of cleared E. coli culture lysatesexpressing SEQ ID NO: 3081 variants with lysate loading of 25 μL lysatein 100 μL reactions and with substrate loading of 1 mM rebaudioside Iand co-substrate loading of 1 mM ADP-glucose. The following reactionconditions were used: 50 mM KPhos buffer, pH 6, 40° C. in a Thermotron®titre-plate shaker with 300 RPM shaking for 18 h. The reactions werediluted 1:4 in water and then quenched by adding 25 μL of the dilutedassay to 75 μL acetonitrile with 0.2% formic acid. The resulting mixturewas precipitated by centrifugation at 4° C. for 10 m. The supernatantswere diluted 1:5 in water and analyzed by LC-MS/MS as described in Table44.1. The resulting 52 engineered variants with glucosyltransferaseactivity on rebaudioside I are listed in Table 50.3.

TABLE 50.3 β1,2GT Round 11 Combinatorial Variants and RebM Levels SEQ IDNO: Amino Acid Differences Increased (nt/aa) (Relative to SEQ ID NO:3082) RebM^(a) 3281/3282 L126Q/P128A/F155R/R161G/N199A + 3283/3284W20Y/P128A/K160S/R161T/G198P + 3285/3286W20Y/L126Q/P128A/K160S/R161T/G198H + 3287/3288L126Q/P128A/K160S/N199A/P369N + 3289/3290 P128A/K160S/R161G/P369N +3291/3292 P128A/F155R/K160S/G198P + 3293/3294L126H/P128A/K160S/G198H/P369N + 3295/3296 P128A/G198H/N199A/P369N +3297/3298 N199A/P369N ++ 3299/3300 L126Q/G198P/P369N + 3301/3302W20Y/L126H/K160S + 3303/3304 L126Q/N199A/P369N + 3305/3306W20Y/F155R/R161T + 3307/3308 W20Y/L126H/F155R/K160S/G198P + 3309/3310F155R/R161G/G198H/N199A + 3311/3312 W20Y/L126H/P128A/R161T + 3313/3314L126H/K160S/G198P/P369N + 3315/3316 L126Q/P128A/K160S/P369N + 3317/3318V121F/L126H/P128A/R161T/P369N ++ 3319/3320 W20Y/P128A/F155R/N199A +3321/3322 P128A/F155R/K160S/R161T/N199A + 3323/3324W20Y/F155R/K160S/R161G + 3325/3326 L126H/K160S/R161G/N199A/P369N ++3327/3328 W20Y/L126Q/P128A/R161T/G198H/N199A + 3329/3330L126H/A196V/G198P/P369N + 3331/3332 P128A/F155R/K160S/R161T ++ 3333/3334W20Y/P128A/F155R/K160S/R161T/G198H + 3335/3336W20Y/L126H/P128A/F155R/R161T ++ 3337/3338 P128A/N199A/P369N ++ 3339/3340R161T/G198P/P369N ++ 3341/3342 W20Y/L126H/K160S/R161T ++ 3343/3344L126H/P128A/R161G/P369N ++ 3345/3346 L126Q/P128A/F155R/K160S/R161T/N199A++ 3347/3348 K160S/R161G/P369N + 3349/3350 K160S/R161T/P369N ++3351/3352 W20Y/P128A/F155R/R161T/N199A ++ 3353/3354L126H/P128A/F155R/K160S/R161T + 3355/3356 W20Y/L126Q/R161G + 3357/3358F155R/N199A ++ 3359/3360 W20Y/L126Q/P128A/K160S/R161T +++ 3361/3362L126H/P128A/R161G/N199A/P369N +++ 3363/3364 P128A/F155R/N199A +++3365/3366 R161T/P369N +++ 3367/3368 L126Q/P128A/K160S/R161T/P369N +++3369/3370 W20Y/L126Q/F155R/R161G +++ 3371/3372 L126Q/P369N +++ 3373/3374W20Y/F155R/R161T/N199A +++ 3375/3376 W20Y/K160S/R161T/G198H +++3377/3378 W20Y/F155R/K160S +++ 3379/3380 L126H/P128A/P369N ++ 3381/3382P128A/P369N ++ 3383/3384 P128A/R161G/N199A/P369N ++ ^(a)Levels ofincreased production were determined relative to the referencepolypeptide of SEQ ID NO: 3082, and defined as follows: “+” = productionat least 3.24-fold, but less than 4.14-fold; “++” = at least 4.14-fold,but less than 6-fold; and “+++” = greater than 6-fold increasedproduction, as compared to the reference polypeptide.Shake Flask Powder Characterization Assay and Analysis for GlucosylTransfer from Sucrose to ADP to Rebaudioside I

A shake flask powder loading dose response experiment was performed tocharacterize activity of the engineered round 11.04 variants onrebaudioside I. Levels of 0.16-5 g/L shake flask powder (SFP) were addedto a 100 μL total reaction volume containing 50 mM potassium phosphatebuffer, pH 6, 1 mM rebaudioside 1, and 1 mM ADP-glucose. The reactionwas performed at 50° C. in a Thermotron® titre-plate shaker at 300 RPMfor 4 h. The reactions were diluted, quenched, and analyzed as describedabove. The production levels of rebaudioside M by the round 11.04variants at 0.3 g/L SFP loading are shown in Table 50.4. All 5 of thevariants listed in Table 50.4 had higher activities than SEQ ID NO: 3082on RebI. The variant with the mutations L126Q, P128A, F155R, K160S,R161T, and N199A (SEQ ID NO: 3346) and its encoding polynucleotide (SEQID NO: 3345) were most improved and were selected for further directedevolution for the glucosylation of rebaudioside I

TABLE 50.4 β1,2GT Round 11 Shake Flask Variants and RebM Levels SEQ IDNO: Amino Acid Differences Increased (nt/aa) (Relative to SEQ ID NO:3082) RebM^(a) 3295/3296 P128A/G198H/N199A/P369N + 3345/3346L126Q/P128A/F155R/K160S/R161T/N199A ++ 3377/3378 W20Y/F155R/K160S ++3381/3382 P128A/P369N ++ 3379/3380 L126H/P128A/P369N ++ ^(a)Levels ofincreased production were determined relative to the referencepolypeptide of SEQ ID NO: 3082, and defined as follows: “+” = productionat least 1.15-fold, but less than 1.8-fold; and “++” = at least 1.8-foldincreased production, as compared to the reference polypeptide.

Example 51 Beta-1,2-ADP-Glycosyltransferase Variants of SEQ ID NO: 3244

In this Example, experiments for evolution and screening of GTpolypeptides derived from SEQ ID NO: 3244 for improved glucosylation ofsteviol glycosides using ADP-glucose are described. Directed evolutionof the GT encoded by SEQ ID NO: 3243 was carried out by constructingcombinatorial libraries of variant genes. Libraries recombined mutationsassociated with improved activity in previous rounds and subjectedcertain structural features of the enzyme with saturation mutagenesis.These libraries were then plated, grown, and screened using the HTPassay described below to provide a twelfth round (“Round 12”) ofengineered GT variant polypeptides with glucosyltransferase activitytoward ADP-glucose and steviol glycosides. Fifty engineered variantswere identified from the recombined beneficial mutations (Table 51.1),and 31 were identified from saturation mutagenesis (Table 51.2).

HTP Coupled Assay for Glucose Transfer from Sucrose to ADP toRebaudioside A

Assays were performed on 96-well plates of cleared E. coli culturelysates expressing SEQ ID NO: 3243 variants. Lysis buffer volume was 400μL, and the lysate was diluted 40-fold. For screening the library inwhich beneficial mutations from previous rounds were recombined, dilutedlysates were preincubated at 62° C. for 0.5 h in an Eppendorfthermocycler. Assays were then conducted with 10 μL diluted lysate and0.15 g/L SUS SFP SEQ ID NO: 1764 in 100 μL reaction volume withsubstrate loading of 15 mM rebaudioside A and co-substrate loadings of0.2 mM ADP (Sigma, >95%) and 37.5 mM sucrose. The following reactionconditions were used: 50 mM potassium phosphate buffer, pH6, 60° C. in aThermotron® titre-plate shaker with 300 RPM shaking for 4.5 h. Thereactions were diluted, quenched, and analyzed as described above. Theresulting engineered variants with glucosyltransferase activity coupledwith SUS SEQ ID NO: 1764 on rebaudioside Aare listed in Table 51.1. Forthe round 12 saturation mutagenesis library, screening was performed asdescribed above, with the exception that the diluted lysates werepreincubated at 65° C. for 0.5 h. The resulting engineered GT variantpolypeptides are listed in Table 51.2. Shake-flask scale cultures weregrown, lysed, and lyophilized to powder for variants listed in Table51.3.

TABLE 51.1 β1,2GT Round 12 Combinatorial Variants and RebD Levels SEQ IDNO: Amino Acid Differences Increased (nt/aa) (Relative to SEQ ID NO:3244) RebD^(a) 3491/3492 K106A/N172R/G200A/E210V/N240E/A242I/ ++E408P/G415A/K416R/N423R 3493/3494 N240E/A242I/G415A/N423R ++ 3495/3496N240E/A242I +++ 3497/3498 V164H/N240E/A242I/G415R/N423R + 3499/3500G200A/N240E/A242I/G407S ++ 3501/3502 K106A/V164H/G200A/E210V/G415A +++3503/3504 N240E +++ 3505/3506 V164H/G200A/E408P/G415A/K416R ++ 3507/3508T409L/G415R + 3509/3510 G200A/E408P/T409L ++ 3511/3512 E210V/N240E +++3513/3514 V164H/N240E/A242I/E408P + 3515/3516K106A/V164H/G200A/N240E/E408P/T409L/ ++ K416R 3517/3518G200A/A242I/G415A/K416R/N423R + 3519/3520K106A/V164H/E210V/N240E/E408P/T409L/ + K416R 3521/3522 N172R/N240E +3523/3524 N172R/N240E/A242I/G415R + 3525/3526 K106A/N240E/A242I/T409L +3527/3528 K106A/V164H/E210V/E408P/T409L + 3529/3530K106A/E210V/N240E/E408P/G415R ++ 3531/3532 K106A/N172R/G200A/E210V/A242I+++ 3533/3534 N240E/E408P +++ 3535/3536 N172R/G200A/E408P/K416R +3537/3538 K106A/N172R/E408P/T409L ++ 3539/3540V164H/N172R/N240E/A242I/G415R ++ 3541/3542 V164H/N172R/A242I/E408P +3543/3544 N172R/G200A/N240E + 3545/3546 N172R/G200A/E210V/N240E/E408P +3547/3548 K106A/N172R/T409L/N423R ++ 3549/3550 N240E/A242I/E408P/K416R +3551/3552 K106A/N240E/A242I ++ 3553/3554 N172R/E210V/G415A ++ 3555/3556V164H/N172R/G200A/E210V/A242I/T409L + 3557/3558 K106A/A242I/E408P/T409L+++ 3559/3560 K106A/N172R/N240E/A242I/T409L/G415R ++ 3561/3562N240E/G415R ++ 3563/3564 K106A/V164H/N172R/A242I/T409L/K416R + 3565/3566G200A/E210V/N240E/A242I/T409L + 3567/3568 V164H/N423R ++ 3569/3570K106A/V164H/G200A/E210V/A242I +++ 3571/3572 K106A/V164H/N240E/G415A ++3573/3574 K106A/G200A/E210V/T409L +++ 3575/3576K106A/V164H/T409L/G415A/N423R + 3577/3578K106A/V164H/N172R/G200A/T409L + 3579/3580 N172R/N240E/T409L + 3581/3582K106A/N172R/A242I/K416R/N423R + 3583/3584 K106A/E210V/N240E + 3585/3586K106A/N172R/A242I + 3587/3588 K106A/N172R/N240E +++ 3589/3590K106A/V164H/N172R/E210V/N240E/G415R + ^(a)Levels of increased productionwere determined relative to the reference polypeptide of SEQ ID NO:3244, and defined as follows: “+” = production at least 1.8-fold, butless than 1.9-fold; “++” = at least 1.9-fold, but less than 2.07-fold;and “+++” = at least 2.07-fold increased production, as compared to thereference polypeptide.

TABLE 51.2 β1,2GT Round 12 Saturation Mutagenesis Variants and RebDLevels SEQ ID NO: Amino Acid Differences Increased (nt/aa) (Relative toSEQ ID NO: 3244) RebD^(a) 3591/3592 C46T + 3593/3594 L49M + 3595/3596L49P +++ 3597/3598 V134S + 3599/3600 K143P +++ 3601/3602 V134A +++3603/3604 L49A ++ 3605/3606 V278L + 3607/3608 V179T + 3609/3610 P232T +3611/3612 C46V +++ 3613/3614 S105A ++ 3615/3616 L49Q ++ 3617/3618 L42I +3619/3620 V179D ++ 3621/3622 I290L ++ 3623/3624 Q35D ++ 3625/3626 L42F++ 3627/3628 V134C ++ 3629/3630 L49S ++ 3631/3632 Q441R + 3633/3634L42V + 3635/3636 A381G + 3637/3638 L401V + 3639/3640 V278I + 3641/3642R14K + 3643/3644 K373R + 3645/3646 Q441I + 3647/3648 S336A +++ 3649/3650M181L +++ 3651/3652 V179A + ^(a)Levels of increased production weredetermined relative to the reference polypeptide of SEQ ID NO: 3244, anddefined as follows: “+” = production at least that of the referencepolypeptide, but less than 1.29-fold; “++” = at least 1.29-fold, butless than 1.4-fold; and “+++” = production at least 1.4-fold increasedproduction, relative to the reference polypeptide.Shake Flask Powder Characterization Assay and Analysis for GlucosylTransfer from Sucrose to ADP to Rebaudioside A

A shake flask powder loading dose response experiment was performed tocharacterize activity of the engineered round 12 variants onrebaudioside A. Levels of 0.006-0.2 g/L shake flask powder (SFP) werepreincubated at 62° C. for 0.5 hand then added to a 100 μL totalreaction volume containing 50 mM potassium phosphate buffer, pH 6, 15 mMrebaudioside A, 0.2 mM ADP, 37.5 mM sucrose, and 0.15 g/L SUS SFP SEQ IDNO: 1764. The reaction was performed at 50° C. and 60° C. in aThermotron® titre-plate shaker at 300 RPM for 4.5 h. The reactions werediluted, quenched, and analyzed as described above. The conversion ofthe round 12 variants on rebaudioside A at 0.025 g/L SFP loading arelisted in Table 51.3. All 7 of the variants listed in Table 51.3 hadhigher activities than SEQ ID NO: 3244 at 60° C., and 4 did not losesignificant activity at 50° C. The variant with the mutations K106A,V164H, G200A, E210V, and G415A (SEQ ID NO: 3502) and its encodingpolynucleotide (SEQ ID NO: 3501) were selected for further directedevolution for the glucosylation of rebaudioside A.

TABLE 51.3 β1,2GT Round 12 Shake Flask Variants and RebD LevelsIncreased SEQ ID NO: Amino Acid Differences RebD, (nt/aa) (Relative toSEQ ID NO: 3244) 60° C.^(a) 3501/3502 K106A/V164H/G200A/E210V/G415A +++3515/3516 K106A/V164H/G200A/N240E/E408P/ + T409L/K416R 3531/3532K106A/N172R/G200A/E210V/A242I ++ 3539/3540 V164H/N172R/N240E/A242I/G415R++ 3573/3574 K106A/G200A/E210V/T409L +++ 3581/3582K106A/N172R/A242I/K416R/N423R ++ 3589/3590K106A/V164H/N172R/E210V/N240E/G415R ++ ^(a)Levels of increasedproduction were determined relative to the reference polypeptide of SEQID NO: 3244, and defined as follows: “+” = production at least1.24-fold, but less than 1.4-fold; “++” = at least 1.4-fold, but lessthan 1.5-fold; and “+++” = at least 1.5-fold increased production,relative to the reference polypeptide.

Example 52 Beta-1,2-ADP-Glycosyltransferase Variants of SEQ ID NO: 3346for Rebaudioside M Production From Rebaudioside I

In this Example, experiments for evolution and screening of GTpolypeptides derived from SEQ ID NO: 3346 for improved glucosylation ofsteviol glycosides are described. Directed evolution of the GT encodedby SEQ ID NO: 3345 was carried out by constructing a library thatsubjected certain structural features of the enzyme to saturationmutagenesis. The library was then plated, grown, and screened using thehigh-throughput (HTP) assay described below to provide a round (“Round12 RebI”) of engineered GT variant polypeptides with glucosyltransferaseactivity toward ADP-glucose and rebaudioside I.

HTP Assay for Glucose Transfer from Sucrose to ADP to Rebaudioside M

Assays were performed on 96-well plates of cleared E. coli culturelysates expressing SEQ ID NO: 3345 variants. Lysis buffer volume was 400μL, and assays were then conducted with 25 L lysate and 0.1 g/L SUS SFPSEQ ID NO: 1804 in 100 μL reaction volume with substrate loading of 1 mMrebaudioside I and co-substrate loadings of 1 mM ADP (Sigma, >95%) and15 mM sucrose. The following reaction conditions were used: 50 mMpotassium phosphate buffer, pH 6, 50° C. in a Thermotron® titre-plateshaker with 300 RPM shaking for 4 h. The reactions were diluted,quenched, and analyzed as described in Example 50. The resultingengineered variants with glucosyltransferase activity coupled with a SuSon rebaudioside I are shown in Table 52.1.

TABLE 52.1 β1,2GT Round 12 RebI Saturation Mutagenesis Variants and RebMLevels SEQ ID NO: Amino Acid Differences Increased (nt/aa) (Relative toSEQ ID NO: 3346) RebM^(a) 3759/3760 G200T + 3761/3762 F152L + 3763/3764P197D + 3765/3766 P197Q ++ 3767/3768 I202W ++ 3769/3770 K143H +++3771/3772 K187S ++ 3773/3774 L195R + 3775/3776 L195Y ++ 3777/3778 G201W++ 3779/3780 Y268Q + 3781/3782 P91M + 3783/3784 G200P ++ 3785/3786P197W + 3787/3788 L21M + 3789/3790 G200E ++ 3791/3792 F125C +++3793/3794 Q127G ++ 3795/3796 G201R +++ 3797/3798 F264S + 3799/3800K143P/A150S + 3801/3802 L21P +++ 3803/3804 A130T/K187Q +++ 3805/3806I202A + 3807/3808 E186V + 3809/3810 G201A ++ 3811/3812 L195S + 3813/3814F264T + 3815/3816 G200R +++ 3817/3818 F156Q + 3819/3820 M364W +3821/3822 L195G ++ 3823/3824 E186N ++ 3825/3826 L145W + 3827/3828 Q365L+++ 3829/3830 G201S ++ 3831/3832 Y268W ++ 3833/3834 L195T + 3835/3836F125M + 3837/3838 M364S + 3839/3840 Y268F ++ 3841/3842 G415D + 3843/3844F125V + 3845/3846 L195F +++ 3847/3848 F156P ++ 3849/3850 P197L +^(a)Levels of increased production were determined relative to thereference polypeptide of SEQ ID NO: 3346, and defined as follows: “+” =production at least 1.07-fold, but less than 1.19-fold; “++” = at least1.19-fold, but less than 1.33-fold; and “+++” = at least 1.33-foldincreased production, as compared to the reference polypeptide.

Example 53 Beta-1,2-ADP-Glycosyltransferase Variants of SEQ ID NO: 3502

In this Example, experiments for evolution and screening of GTpolypeptides derived from SEQ ID NO: 3502 for improved glucosylation ofsteviol glycosides using ADP-glucose are described. Directed evolutionof the GT encoded by SEQ ID NO: 3501 was carried out by constructingcombinatorial libraries of variant genes. Libraries recombined mutationsassociated with improved activity in previous rounds and subjectedcertain structural features of the enzyme with saturation mutagenesis.These libraries were then plated, grown, and screened using the HTPassay described below to provide a thirteenth round (“Round 13”) ofengineered GT variant polypeptides with glucosyltransferase activitytoward ADP-glucose and steviol glycosides. 53 engineered variants wereidentified from the recombined beneficial mutations (Table 53.1), and 24were identified from saturation mutagenesis (Table 53.2).

HTP Coupled Assay for Glucose Transfer from Sucrose to ADP toRebaudioside A

Assays were performed on 96-well plates of cleared E. coli culturelysates expressing SEQ ID NO: 3501 variants. Lysis buffer volume was 400μL, and the lysate was diluted 40-fold. For screening the library inwhich beneficial mutations from previous rounds were recombined, dilutedlysates were preincubated at 69° C. for 0.8 h in an Eppendorfthermocycler. Assays were then conducted with 10 μL diluted lysate and0.15 g/L SUS SFP SEQ ID NO: 1764 in 100 μL reaction volume withsubstrate loading of 15 mM rebaudioside A and co-substrate loadings of0.2 mM ADP (Sigma, >95%) and 37.5 mM sucrose. The following reactionconditions were used: 50 mM potassium phosphate buffer, pH 6, 60° C. ina Thermotron® titre-plate shaker with 300 RPM shaking for 4 h. Thereactions were diluted, quenched, and analyzed as described above. Theresulting engineered variants with glucosyltransferase activity coupledwith SUS SEQ ID NO: 1764 on rebaudioside A are listed in Table 53.1. Forthe round 13 saturation mutagenesis library, screening was performed asdescribed above with the exceptions that the diluted lysates werepreincubated at 68° C. for 0.5 h, 0.1 g/L SUS SFP SEQ ID NO: 1804 wasused, and either 0.1 or 0.2 mM ADP was used. The resulting engineered GTvariant polypeptides are listed in Table 53.2. Shake-flask scalecultures were grown, lysed, and lyophilized to powder for the variantslisted in Table 53.3.

TABLE 53.1 β1,2GT Round 13 Combinatorial Variants and RebD Levels SEQ IDNO: Amino Acid Differences Increased (nt/aa) (Relative to SEQ ID NO:3502) RebD^(a) 3653/3654 P237L + 3655/3656 E186G/P237V/N286S + 3657/3658H2-/H5N/L113I/E186G/P237V/T406Q +++ 3659/3660H5N/E186G/P237V/K245T/E2561/T406Q + 3661/3662 E186G/P237L ++ 3663/3664K34R/L113I/E186G/P237L/T406N + 3665/3666 H5N/K34R/H73A/L113I/T406N +3667/3668 H5N/E186G/P237V/T406Q ++ 3669/3670 H73A/P237V + 3671/3672H5N/L113I/P237V/T406N + 3673/3674 E186G ++ 3675/3676H2-/H73A/E186G/P237V + 3677/3678 H73A/E186G/P237L/T406Q +++ 3679/3680H5N/E256L/T406Q ++ 3681/3682 L113I/E186G/T406Q + 3683/3684 E186G/T406N +3685/3686 E186G/T406Q + 3687/3688 H5N/H73A/E186G/T406N ++ 3689/3690P189S/N333H/F355A/S421Q + 3691/3692 H2S/N286Q/F355A/K416R/K422R ++3693/3694 H2S/P189S/N286Q + 3695/3696 H73P/N172R/N240E/A242I/E408P +3697/3698 H73P/Q235M/N240E/E408P + 3699/3700H73P/N172R/Q235M/T239A/N240E ++ 3701/3702 H73P/N172R/T239A/N240E +3703/3704 H73P/T239A/A242I ++ 3705/3706 T239A/N240E + 3707/3708N172R/Q235M/N240E + 3709/3710 L72Y/N172R/N240E/A242I + 3711/3712T239A/N240E/A242I/E256S/D323P + 3713/3714 N240E/A242I/E256S/E438T +3715/3716 N172R/E188G/D323P ++ 3717/3718 H73P/Q235M/N240E/D323P ++3719/3720 A242I/E408P + 3721/3722 T239A/N240E/E408P +++ 3723/3724H73P/N172R + 3725/3726 H73P/N172R/T239A/N240E/A242I +++ 3727/3728T239A/A242I/E408P + 3729/3730 L72Y/H73P/N172R/Q235M/N240E/A242I/ ++E438T 3731/3732 Q235M/T239A/N240E ++ 3733/3734 N172R + 3735/3736Q235M/N240E/A242I +++ 3737/3738 N172R/T239A/N240E/A242I/D323P/E408P ++3739/3740 H73P/N172R/Q235M/T239A/A242I + 3741/3742 N172R/Q235M ++3743/3744 T239A/N240E/A242I/D323P/E408P ++ 3745/3746H73P/N172R/N240E/E408P +++ 3747/3748 L72Y/N172R/T239A/N240E/A242I/E408P+++ 3749/3750 N172R/Q235M/T239A/E408P + 3751/3752N172R/N240E/E299A/D323P ++ 3753/3754 N172R/Q235M/N240E/A242I/E438T +++3755/3756 N172R/N240E +++ 3757/3758 N172R/Q235M/T239A/N240E/A242I +++^(a)Levels of increased production were determined relative to thereference polypeptide of SEQ ID NO: 3502, and defined as follows: “+” =production at least 1.58-fold, but less than 1.79-fold; “++” = at least1.79-fold, but less than 2.07-fold; and “+++” = at least 2.07-foldincreased production, as compared to the reference polypeptide.

TABLE 53.2 β1,2GT Round 13 Saturation Mutagenesis Variants and RebDLevels SEQ ID NO: Amino Acid Differences Increased (nt/aa) (Relative toSEQ ID NO: 3502) RebD^(a) 3851/3852 E186R ++ 3853/3854 K187T ++3855/3856 N199Y + 3857/3858 Q132H ++ 3859/3860 S153G + 3861/3862 Q132T +3863/3864 A196S + 3865/3866 S153A + 3867/3868 Q127V +++ 3869/3870N199A + 3871/3872 I144V + 3873/3874 E186A ++ 3875/3876 K187A + 3877/3878Q127L + 3879/3880 Q132K + 3881/3882 G96P ++ 3883/3884 K187R +++3885/3886 N199S ++ 3887/3888 E186G +++ 3889/3890 G96A ++ 3891/3892A200S + 3893/3894 F156W + 3895/3896 Q127I +++ 3897/3898 F155M +++^(a)Levels of increased production were determined relative to thereference polypeptide of SEQ ID NO: 3502, and defined as follows: “+” =production at least that of the reference polypeptide, but less than1.15-fold; “++” = at least 1.15-fold, but less than 1.18-fold; and “+++”= at least 1.18-fold increased production, as compared to the referencepolypeptide.Shake Flask Powder Characterization Assay and Analysis for GlucosylTransfer from Sucrose to ADP to Rebaudioside A

A shake flask powder loading dose response experiment was performed tocharacterize activity of the engineered round 13 variants onrebaudioside A. Levels of 0.006-0.2 g/L shake flask powder (SFP) werepreincubated at 69° C. for 50 min and then added to a 100 μL totalreaction volume containing 50 mM potassium phosphate buffer, pH6, 15 mMrebaudioside A, 0.2 mM ADP, 37.5 mM sucrose, and 0.1 g/L SUS SFP SEQ IDNO: 1804. The reaction was performed at 60° C. in a Thermotron®titre-plate shaker at 300 RPM for 4h. The reactions were diluted,quenched, and analyzed as described above. The conversion of the round13 variants on rebaudioside A at 0.025 g/L SFP loading are listed inTable 53.3. All 10 of the variants listed in Table 53.3 had higheractivities than SEQ ID NO: 3502. The variant with the mutations H73P,N172R, N240E, A242I, and E408P (SEQ ID NO: 3696) and its encodingpolynucleotide (SEQ ID NO: 3695) were the most improved and wereselected for further directed evolution for the glucosylation ofrebaudioside A.

TABLE 53.3 β1,2GT Round 13 Shake Flask Variants and RebD Levels SEQ IDNO: Amino Acid Differences Increased (nt/aa) (Relative to SEQ ID NO:3502) RebD^(a) 3695/3696 H73P/N172R/N240E/A242I/E408P +++ 3689/3690P189S/N333H/F355A/S421Q +++ 3675/3676 H2-/H73A/E186G/P237V ++ 3745/3746H73P/N172R/N240E/E408P ++ 3691/3692 H2S/N286Q/F355A/K416R/K422R ++3657/3658 H2-/H5N/L113I/E186G/P237V/T406Q + 3699/3700H73P/N172R/Q235M/T239A/N240E + 3703/3704 H73P/T239A/A242I + 3693/3694H2S/P189S/N286Q + 3677/3678 H73A/E186G/P237L/T406Q + ^(a)Levels ofincreased production were determined relative to the referencepolypeptide of SEQ ID NO: 3502 and defined as follows: “+” = productionat least 1.79-fold that of the reference polypeptide, but less than3-fold; “++” = at least 3-fold increased production but less than3.6-fold; and “+++” = at least 3.6-fold increased production relative toreference polypeptide.

Example 54 Beta-1,2-ADP-Glycosyltransferase Variants of SEQ ID NO: 3696

In this Example, experiments for evolution and screening of GTpolypeptides derived from SEQ ID NO: 3696 for improved glucosylation ofsteviol glycosides using ADP-glucose are described. Directed evolutionof the GT encoded by SEQ ID NO: 3695 was carried out by constructingcombinatorial libraries of variant genes. Libraries recombined mutationsassociated with improved activity in previous rounds and subjectedcertain structural features of the enzyme with saturation mutagenesis.These libraries were then plated, grown, and screened using the HTPassay described below to provide a fourteenth round (“Round 14”) ofengineered GT variant polypeptides with glucosyltransferase activitytoward ADP-glucose and steviol glycosides. Seventy-five engineeredvariants were identified from the recombined beneficial mutations (Table54.1), and 49 were identified from saturation mutagenesis (Table 54.2).

HTP Coupled Assay for Glucose Transfer from Sucrose to ADP toRebaudioside A

Assays were performed on 96-well plates of cleared E. coli culturelysates expressing SEQ ID NO: 3695 variants. Lysis buffer volume was 400μL, and the lysate was diluted 40-fold. For screening the library inwhich beneficial mutations from previous rounds were recombined, dilutedlysates were preincubated at 72 for 0.5 in an Eppendorf thermocycler.Assays were then conducted with 10 μL diluted lysate and 0.1 g/L sucrosesynthetase (SUS) SFP SEQ ID NO:1804 in 100 μL reaction volume withsubstrate loading of 20 mM rebaudioside A and co-substrate loadings of0.2 mM ADP (Sigma, >95%) and 50 mM sucrose. The following reactionconditions were used: 50M potassium phosphate buffer, pH6, 60° C. in aThermotron® titre-plate shaker with 300 RPM shaking for 4h. Thereactions were diluted, quenched, and analyzed as described above. Theresulting engineered variants with glucosyltransferase activity coupledwith a SuS on rebaudioside Aare listed in Table 54.1. For the round 14saturation mutagenesis library, screening was performed as describedabove with the exceptions that the lysate was diluted 80-fold and waspreincubated at 70° C. for 0.5 h. The resulting engineered GT variantpolypeptides are listed in Table 54.2. Shake-flask scale cultures weregrown, lysed, and lyophilized to powder for variants listed in Table54.3.

TABLE 54.1 β1,2GT Round 14 Combinatorial Variants and RebD Levels SEQ IDNO: Amino Acid Differences Increased (nt/aa) (Relative to SEQ ID NO:3696) RebD^(a) 3899/3900 I26V/L42V/C46V/V134A + 3901/3902L42V/C46V/L49P/V134C/Q235R/F355A/ ++ L401V 3903/3904L42I/C46V/L49S/V134C/E186G + 3905/3906 I26V/L49Q/V134A + 3907/3908C46V/L49Q/V134T + 3909/3910 I26V/V134T/L401V +++ 3911/3912I26V/L42V/V134C/L401V + 3913/3914 L42V/C46V/L49A/V134A/L401V + 3915/3916I26V/L42I/L49Q/V134C/E186G/F355A +++ 3917/3918I26V/L42I/L49Q/V134C/L401V ++ 3919/3920 I26V/L42V/L49S/V134C + 3921/3922L42V/L49A/V134C/E186G/F355A +++ 3923/3924I26V/L42V/C46V/L49A/V134C/E186G + 3925/3926L42V/C46V/L49S/T97P/V134C/L401V ++ 3927/3928L42V/C46V/L49A/V134T/E186G/F355A + 3929/3930I26V/V134A/E186G/F355A/L401V + 3931/3932 L42I/C46V/L49S/E186G +3933/3934 I26V/L42V/C46V/L49A ++ 3935/3936 I26V/F355A/L401V + 3937/3938L42V/V134C/E186G ++ 3939/3940 L42I/C46V/L49A/V134A ++ 3941/3942L42V/L49S/V134A/E186G/L401V ++ 3943/3944 E186G/F355A + 3945/3946I26V/L42V ++ 3947/3948 V134C/L401V + 3949/3950L42V/L49S/V134C/F355A/L401V + 3951/3952 I26V/V134A +++ 3953/3954I26V/V134C/F355A + 3955/3956 I26V/L42V/C46V/L49A/V134A +++ 3957/3958L49S/V134C/F355A + 3959/3960 L42V/E186G/F355A ++ 3961/3962H2-/K143P/P232T/K373R/Q441R + 3963/3964 H2-/K143P/K373R/Q441R +3965/3966 H5N/P237L/K373R/K422R + 3967/3968 S105A/K373R/Q441R +3969/3970 H5N/K143P/P189S/P232T/P237V/Q441R + 3971/3972 K143P/P232T +3973/3974 K143P/P189S/P232T/K422R/Q441R + 3975/3976H5N/K143P/P232T/K373R/K422R/Q441R + 3977/3978S105A/K143P/P232T/P237V/K373R/K416R/ + K422R/Q441R 3979/3980K143P/P237V + 3981/3982 H2-/H5N/Q35D/S105A/K143P/P237V/K373R/ +K416R/K422R 3983/3984 P1895/P232T/K373R/K416R/K422R/Q441R ++ 3985/3986S105A/P237L/K373R/K422R/Q441R +++ 3987/3988 S105A/K373R +++ 3989/3990H5N/S105A/P237V/K416R/K422R/Q441R + 3991/3992H2-/H5N/Q35D/P232T/V278L/K373R/K416R ++ 3993/3994 S105A/P1895/P237V ++3995/3996 H2-/H5N/S105A/K143P/P232T/K373R/ + K416R/K422R 3997/3998H2-/H5N/V278L +++ 3999/4000 P232T/P237L ++ 4001/4002 P237L/K422R +4003/4004 H5N/K143P/K373R +++ 4005/4006 K143P +++ 4007/4008Q35D/S105A/P189S/P232T/K373R/K416R + 4009/4010H2-/H5N/Q35D/K143P/P232T/P237L/K416R/ + K422R/Q441R 4011/4012S105A/P237L/V278L/K373R/K416R/K422R ++ 4013/4014H5N/S105A/P232T/P237L/K373R/K416R/ ++ Q441R 4015/4016H5N/K143P/P232T/P237L/K416R/K422R +++ 4017/4018 S105A/K143P/V278L/K373R++ 4019/4020 K143P/P237L/K422R/Q441R +++ 4021/4022H5N/P232T/K416R/K422R + 4023/4024 H5N/Q35D/P232T/K373R/K416R/K422R ++4025/4026 P189S/P237L/V2781/K416R/Q441R ++ 4027/4028H5N/K143P/P237L/V278L/K373R/K416R/ ++ K422R 4029/4030K143P/P189S/P237L/K373R/K416R/K422R +++ 4031/4032 K143P/P1895 +++4033/4034 H5N/S105A/P237L/V278I/K422R/Q441R + 4035/4036 H5N/K373R/K416R++ 4037/4038 P237V/K373R/Q441R ++ 4039/4040 Q35D + 4041/4042H2-/S105A/K143P/P232T/P237L/V278L/ ++ K373R/Q441R 4043/4044H5N/P1895/P237V/V2781/K373R/K416R/ + K422R/Q441R 4045/4046H5N/K143P/P232T/P237L/V278L ++ 4047/4048 P237V/K373R + ^(a)Levels ofincreased production were determined relative to the referencepolypeptide of SEQ ID NO: 3696, and defined as follows: “+” = productionat least 1.5-fold, but less than 1.75-fold; “++” = at least 1.75-fold,but less than 2-fold; and “+++” = at least 2-fold increased production,as compared to the reference polypeptide.

TABLE 54.2 β1,2GT Round 14 Saturation Mutagenesis Variants and RebDLevels SEQ ID NO: Amino Acid Differences Increased (nt/aa) (Relative toSEQ ID NO: 3696) RebD^(a) 4049/4050 A426L + 4051/4052 S352A ++ 4053/4054T239F + 4055/4056 R176Q +++ 4057/4058 Q235R +++ 4059/4060 F158W +4061/4062 H330C ++ 4063/4064 T239Y ++ 4065/4066 D243Y +++ 4067/4068T239P ++ 4069/4070 H164E +++ 4071/4072 D249H + 4073/4074 D243R +4075/4076 H3N + 4077/4078 R427S + 4079/4080 H164F + 4081/4082 I50P +4083/4084 S8R + 4085/4086 T239G + 4087/4088 T239M ++ 4089/4090 S8V ++4091/4092 D249G ++ 4093/4094 D243G ++ 4095/4096 P237A + 4097/4098 D243H++ 4099/4100 D243S ++ 4101/4102 H164L + 4103/4104 P237L + 4105/4106D193F + 4107/4108 R176P +++ 4109/4110 150V +++ 4111/4112 R176T +4113/4114 H164A + 4115/4116 A101L ++ 4117/4118 I248T + 4119/4120 T223A++ 4121/4122 T223L/D243G + 4123/4124 Q137R ++ 4125/4126 R427T ++4127/4128 E61Y + 4129/4130 E240V +++ 4131/4132 E61N +++ 4133/4134M364Q + 4135/4136 Q301A ++ 4137/4138 K62A ++ 4139/4140 R161L + 4141/4142D323T + 4143/4144 D244N +++ 4145/4146 T239Q + ^(a)Levels of increasedproduction were determined relative to the reference polypeptide of SEQID NO: 3696, and defined as follows: “+” = production at least 1.1-fold,but less than 1.18-fold; “++” = at least 1.18-fold, but less than1.24-fold; and “+++” = at least 1.24-fold increased production, ascompared to the reference polypeptide.Shake Flask Powder Characterization Assay and Analysis for GlucosylTransfer from Sucrose to ADP to Rebaudioside A

A shake flask powder loading dose response experiment was performed tocharacterize activity of the engineered round 14 variants onrebaudioside A. Levels of 0.006-0.2 g/L shake flask powder (SFP) werepreincubated at 72° C. for 0.5 hand then added to a 100 μL totalreaction volume containing 50 mM potassium phosphate buffer, pH 6, 20 mMrebaudioside A, 0.2 mM ADP, 50 mM sucrose, and 0.1 g/L SUS SFP SEQ IDNO: 1804. The reaction was performed at 60° C. with preincubated SFP orat 55° C. with SFP that had not undergone preincubation in a Thermotron®titre-plate shaker at 300 RPM for 4h. The reactions were diluted,quenched, and analyzed as described above. The production levels ofrebaudioside D by the round 14 variants at 0.05 g/L SFP loading areshown in Table 54.3. Three out of the 8 variants listed in Table 54.3had higher activities under both conditions than SEQ ID NO: 3696. Thevariant with the mutations I26V, L42V, C46V, L49A, and V134A (SEQ ID NO:3956) and its encoding polynucleotide (SEQ ID NO: 3955) were the mostimproved taking into account both conditions and were selected forfurther directed evolution for the glucosylation of rebaudioside A.

TABLE 54.3 β1,2GT Round 14 Shake Flask Variants and RebD LevelsIncreased SEQ ID NO: Amino Acid Differences RebD, (nt/aa) (Relative toSEQ ID NO: 3696) 60° C.^(a) 3899/3900 I26V/L42V/C46V/V134A ++ 3921/3922L42V/L49A/V134C/E186G/F355A ++ 3955/3956 I26V/L42V/C46V/L49A/V134A ++3961/3962 H2-/K143P/P232T/K373R/Q441R + 3969/3970H5N/K143P/P189S/P232T/P237V/Q441R + 3987/3988 S105A/K373R + 3997/3998H2-/H5N/V278L + 4041/4042 H2-/S105A/K143P/P232T/P237L/V278L/ +K373R/Q441R ^(a)Levels of increased production were determined relativeto the reference polypeptide of SEQ ID NO: 3696, and defined as follows:“+” = production at least 1.13-fold, but less than 1.4-fold; and “++” =at least 1.4-fold increased production, as compared to the referencepolypeptide.

Example 55 Beta-1,2-ADP-Glycosyltransferase Variants of SEQ ID NO: 3956

In this Example, experiments for evolution and screening of GTpolypeptides derived from SEQ ID NO: 3956 for improved glucosylation ofsteviol glycosides using ADP-glucose are described. Directed evolutionof the GT encoded by SEQ ID NO: 3955 was carried out by constructingcombinatorial libraries of variant genes. Libraries recombined mutationsassociated with improved activity in previous rounds and subjectedcertain structural features of the enzyme with saturation mutagenesis.These libraries were then plated, grown, and screened using the HTPassay described below, to provide a fifteenth round (“Round 15”) ofengineered GT variant polypeptides with glucosyltransferase activitytoward ADP-glucose and steviol glycosides. Sixty-two engineered variantswere identified from the recombined beneficial mutations (Table 55.1),and 113 were identified from saturation mutagenesis (Table 55.2).

HTP Coupled Assay for Glucose Transfer from Sucrose to ADP toRebaudioside A

Assays were performed on 96-well plates of cleared E. coli culturelysates expressing SEQ ID NO: 3956 variants. Lysis buffer volume was 400μL, and the lysate was diluted 40-fold. For screening the library inwhich beneficial mutations from previous rounds were recombined, dilutedlysates were preincubated at 76 for 0.5h in an Eppendorf thermocycler.Assays were then conducted with 10 μL diluted lysate and 0.1 g/L SUS SFPSEQ ID NO: 1840 in 100 μL reaction volume with substrate loading of 20mM rebaudioside A and co-substrate loadings of 0.2 mM ADP (Sigma, >95%)and 50 mM sucrose. The following reaction conditions were used: 50 mMpotassium phosphate buffer, pH6, 60° C. in a Thermotron® titre-plateshaker with 300 RPM shaking for 4h. The reactions were diluted,quenched, and analyzed as described above. The resulting engineeredvariants with glucosyltransferase activity coupled with SUS SEQ ID NO:1840 on rebaudioside Aare listed in Table 55.1. For the round 15saturation mutagenesis library, screening was performed as describedabove with the exceptions that the diluted lysates were preincubated at65° C. for 16 hand 0.1 g/L SUS SFP SEQ ID NO: 2064 was used. Theresulting engineered GT variant polypeptides are listed in Table 55.2.Shake-flask scale cultures were grown, lysed, and lyophilized to powderfor the variants listed in Table 55.3.

TABLE 55.1 β1,2GT Round 15 Combinatorial Variants and RebD Levels SEQ IDNO: Amino Acid Differences Increased (nt/aa) (Relative to SEQ ID NO:3956) RebD^(a) 4147/4148 A49S/S153A/F155M ++ 4149/4150S153A/N199S/T406Q + 4151/4152 G96A/S153A/F155M/F156W + 4153/4154G96P/Q127I/S153A/F155M + 4155/4156 A49S/G96P/Q132K/F155M/P237L/V278L +4157/4158 Q132H/F155M + 4159/4160 A49S/G96P/F155M/N199S/A200S/T406Q +4161/4162 G96P/Q127I/Q132H/S153A/V278L ++ 4163/4164G96P/S153A/F155M/N199S/A200S/P237L + 4165/4166S153A/F155M/A196S/N199S/P237L + 4167/4168Q127I/Q132H/F155M/F156W/T406Q + 4169/4170 G96A/Q132K/S153A/F155M/F156W +4171/4172 G96A/Q132K/S153A/F155M/F156W/A200S + 4173/4174G96P/S153A/T406Q + 4175/4176 Q127I/T406Q + 4177/4178 Q132K/P237L +4179/4180 A49S/G96A/Q127I/Q132K/F156W/A196S +++ 4181/4182A49S/G96P/Q127I/S153A/V278L +++ 4183/4184Q127I/S153A/F155M/N199S/A200S/P237L ++ 4185/4186 Q127I/Q132H/S153A/F156W+++ 4187/4188 S153A/P237L ++ 4189/4190 Q132K/S153A/F155M +++ 4191/4192A49S/G96P/Q132H/F155M ++ 4193/4194 S153A/F155M/F156W/P237L +++ 4195/4196G96A/Q132H/S153A/F156W +++ 4197/4198 G96P/Q132H/S153A/F155M/F156W ++4199/4200 A49S/G96A/Q132H/S153A/N199S/A200S + 4201/4202 G96P/Q132K/V278L+++ 4203/4204 Q127I/Q132H + 4205/4206 G96P/Q132H/A196S/N199S + 4207/4208G96P/Q132K/S153A/F155M ++ 4209/4210 Q132H/S153A/P237L/T406Q ++ 4211/4212F155M/N1995 ++ 4213/4214 A495/G96P/Q132H/S153A/F155M/F156W/ ++P237L/V278L/T406Q 4215/4216 H5N/P73A/I144V/K187R/K373R/K422R ++4217/4218 H5N/I144V/V179T/M181L/K373R/K422R ++ 4219/4220V179T/M181L/E186A + 4221/4222 K143P/V179T/M181L/E186G/K187R/K422R/ +N423R 4223/4224 V179D/K187T/K373R/K422R ++ 4225/4226H5N/P73A/I144V/V179D/E186A/K373R/ +++ N423R 4227/4228I144V/V179T/M181L/E186A/K187R/K373R +++ 4229/4230V179D/M181L/E186G/K187R/N423R ++ 4231/4232P73A/L113I/V179T/M181L/E186A/K187R ++ 4233/4234 K373R/N423R + 4235/4236V179T/E186A/K187T + 4237/4238 M181L/E186G/K187R/K422R/N423R + 4239/4240L113I/I144V/E186G/N423R + 4241/4242 I144V/V179T/E186A/K187T/K373R ++4243/4244 K143P/I144V/V179T/M181L/E186G/K187R/ + K422R 4245/4246L113I/K373R/K422R + 4247/4248 P73A/L113I/K143P/V179T/M181L/K422R ++4249/4250 L113I/I144V/K373R ++ 4251/4252 P73A/M181L/E186A/K187T +++4253/4254 H5N/K143P/I144V/V179T/M181L/E186G/ + K187R/K373R 4255/4256P73A/K143P/I144V/V179D/E186G/K187T/ +++ K373R/N423R 4257/4258H5N/I144V/K373R/K422R +++ 4259/4260 L113I/K143P/V179D/E186G/K187R +4261/4262 P73A/V179D/M181L/E186G/K187R/K373R ++ 4263/4264L113I/M181L/E186A/K373R/K422R + 4265/4266H5N/P73A/K143P/I144V/V179D/E186G/ + K187R/K422R 4267/4268P73A/V179D/M181L/E186A/K373R/K422R ++ 4269/4270H5N/P73A/L113I/E186G/K187R/K373R/ + N423R ^(a)Levels of increasedproduction were determined relative to the reference polypeptide of SEQID NO: 3956, and defined as follows: “+” = production at least1.75-fold, but less than 2-fold; “++” = at least 2-fold, but less than2.38-fold; and “+++” = at least 2.38-fold increased production, ascompared to the reference polypeptide.

TABLE 55.2 β1,2GT Round 15 Saturation Mutagenesis Variants and RebDLevels SEQ ID NO: Amino Acid Differences Increased (nt/aa) (Relative toSEQ ID NO: 3956) RebD^(a) 4271/4272 T12L + 4273/4274 L222F ++ 4275/4276T220G +++ 4277/4278 M238I + 4279/4280 K117N + 4281/4282 E388S +4283/4284 K57H + 4285/4286 F41H + 4287/4288 S66L + 4289/4290 Y449L +++4291/4292 P73S ++ 4293/4294 L74M/M238V + 4295/4296 D236L ++ 4297/4298H83R + 4299/4300 N65R + 4301/4302 P408T ++ 4303/4304 E388A + 4305/4306K214T + 4307/4308 M238G +++ 4309/4310 Q132G ++ 4311/4312 P82S +4313/4314 D387P ++ 4315/4316 Y449H ++ 4317/4318 M238A ++ 4319/4320 K450V++ 4321/4322 K309T +++ 4323/4324 K57G ++ 4325/4326 D389G ++ 4327/4328K103Q + 4329/4330 R391A +++ 4331/4332 Y44H + 4333/4334 K160Q + 4335/4336T406S + 4337/4338 R391L + 4339/4340 T406G ++ 4341/4342 G138K + 4343/4344T220S ++ 4345/4346 D236T + 4347/4348 M238S + 4349/4350 Y44S/K187T ++4351/4352 N65Q ++ 4353/4354 N286G + 4355/4356 R391V + 4357/4358 R418H +4359/4360 H85W + 4361/4362 L135E + 4363/4364 G138T + 4365/4366 P140G +4367/4368 L45Y +++ 4369/4370 Q11L + 4371/4372 R418A +++ 4373/4374K214H + 4375/4376 T223D +++ 4377/4378 V70W + 4379/4380 M238R +++4381/4382 H393P ++ 4383/4384 N65G +++ 4385/4386 N65C ++ 4387/4388 I55G +4389/4390 E299V + 4391/4392 D236V ++ 4393/4394 K103H + 4395/4396 R182I++ 4397/4398 P408L + 4399/4400 K57P ++ 4401/4402 D389V ++ 4403/4404Q114I ++ 4405/4406 K111F + 4407/4408 Y449S + 4409/4410 K309R ++4411/4412 R182L +++ 4413/4414 I55L + 4415/4416 K160T + 4417/4418 K117S +4419/4420 T12I + 4421/4422 H393T + 4423/4424 R418M ++ 4425/4426 Y449R +4427/4428 K422L ++ 4429/4430 K226Q + 4431/4432 E430P + 4433/4434 I55A ++4435/4436 K56R ++ 4437/4438 K450N + 4439/4440 N65P + 4441/4442 L113S +++4443/4444 P162L +++ 4445/4446 K160D +++ 4447/4448 D387R +++ 4449/4450E256R +++ 4451/4452 P73H +++ 4453/4454 P167L + 4455/4456 P140C +++4457/4458 I412G +++ 4459/4460 L159N ++ 4461/4462 I412R ++ 4463/4464R391S +++ 4465/4466 Q114G + 4467/4468 M238T ++ 4469/4470 L72V +++4471/4472 G138S ++ 4473/4474 Q11D ++ 4475/4476 R182Y + 4477/4478 D389A +4479/4480 K309S + 4481/4482 E429D ++ 4483/4484 R182Q + 4485/4486 D387Q +4487/4488 K309P ++ 4489/4490 D389L + 4491/4492 R182V + 4493/4494 Y449G +4495/4496 R182T +++ ^(a)Levels of increased production were determinedrelative to the reference polypeptide of SEQ ID NO: 3956, and defined asfollows: “+” = production at least 1.15-fold, but less than 1.22-fold;“++” = at least 1.22-fold, but less than 1.35-fold; and “+++” = at least1.35-fold increased production, as compared to the referencepolypeptide.Shake Flask Powder Characterization Assay and Analysis for GlucosylTransfer from Sucrose to ADP to Rebaudioside A

A shake flask powder loading dose response experiment was performed tocharacterize activity of the engineered round 15 variants onrebaudioside A. Levels of 0.006-0.2 g/L shake flask powder (SFP) werepreincubated at 76° C. for 0.5 hand then added to a 100 μL totalreaction volume containing 50 mM potassium phosphate buffer, pH6, 20 mMrebaudioside A, 0.2 mM ADP, 50 mM sucrose, and 0.1 g/L SUS SFP SEQ IDNO: 2064. The reaction was performed at 60° C. with preincubated SFP orat 55° C. without preincubation in a Thermotron® titre-plate shaker at300 RPM for 4h. The reactions were diluted, quenched, and analyzed asdescribed above. The production of rebaudioside D by the round 15variants at 0.025 g/L SFP loading are listed in Table 55.3. Five out ofthe 9 variants listed in Table 55.3 had higher activities under bothconditions than SEQ ID NO: 3956. The variant with the mutations P73A,K143P, I144V, V1791D, E186G, K187T, K373R, and N423R (SEQ ID NO: 4256)and its encoding polynucleotide (SEQ ID NO: 4255) were the most improvedtaking into account both conditions and were selected for furtherdirected evolution for the glucosylation of rebaudioside A.

TABLE 55.3 β1, 2GT Round 15 Shake Flask Variants and RebD Levels SEQ IDNO: Increased (nt/aa) Amino Acid Differences (Relative to SEQ ID NO:3956) RebD, 60° C^(a) 4149/4150 S153A/N199S/T406Q ++ 4151/4152G96A/S153A/F155M/F156W + 4193/4194 S153A/F155M/F156W/P237L + 4217/4218H5N/I144V/V179T/M181L/K373R/K422R ++ 4225/4226H5N/P73A/I144V/V179D/E186A/K373R/N423R +++ 4251/4252P73A/M181L/E186A/K187T + 4255/4256P73A/K143P/I144V/V179D/E186G/K187T/K373R/N423R +++ 4267/4268P73A/V179D/M181L/E186A/K373R/K422R + ^(a)Levels of increased productionwere determined relative to the reference polypeptide of SEQ ID NO:3956, and defined as follows: “+” = production at least 1.4-fold, butless than 1.75-fold; “++” = at least 1.75-fold, but less than 1.9-fold;and “+++” = at least 1.9-fold increased production, as compared to thereference polypeptide.

Example 56 Beta-1,2-ADP-Glycosyltransferase Variants of SEQ ID NO: 4256

In this Example, experiments for evolution and screening of GTpolypeptides derived from SEQ ID NO: 4256 for improved glucosylation ofsteviol glycosides using ADP-glucose are described. Directed evolutionof the GT encoded by SEQ ID NO: 4255 was carried out by constructingcombinatorial libraries of variant genes. Libraries recombined mutationsassociated with improved activity in previous rounds and subjectedcertain structural features of the enzyme with saturation mutagenesis.These libraries were then plated, grown, and screened using the HTPassay described below to provide a sixteenth round (“Round 16”) ofengineered GT variant polypeptides with glucosyltransferase activitytoward ADP-glucose and steviol glycosides. Twenty-seven engineeredvariants were identified from the recombined beneficial mutations (Table56.1), and 66 were identified from saturation mutagenesis (Table 56.2).

HTP Coupled Assay for Glucose Transfer from Sucrose to ADP toRebaudioside A

Assays were performed on 96-well plates of cleared E. coli culturelysates expressing SEQ ID NO: 4255 variants. Lysis buffer volume was 400μL, and the lysate was diluted 40-fold. For screening the library inwhich beneficial mutations from previous rounds were recombined, dilutedlysates were preincubated at 79° C. for 0.5 h in an Eppendorfthermocycler. Assays were then conducted with 10 μL diluted lysate and0.05 g/L sucrose synthetase (SUS) SFP SEQ ID NO: 2064 in 100 μL reactionvolume with substrate loading of 20 mM rebaudioside A and co-substrateloadings of 0.2 mM ADP (Sigma, >95%) and 50 mM sucrose. The followingreaction conditions were used: 50 mM potassium phosphate buffer, pH 6,60° C. in a Thermotron® titre-plate shaker with 300 RPM shaking for 4h.The reactions were diluted, quenched, and analyzed as described above.The resulting engineered variants with glucosyltransferase activitycoupled with SUS SEQ ID NO: 2064 on rebaudioside A are listed in Table56.1. For the round 16 saturation mutagenesis library, screening wasperformed as described above with the exception that 0.05 g/L sucrosesynthetase SFP SEQ ID NO: 2510 was used. The resulting engineered GTvariant polypeptides are listed in Table 56.2. Shake-flask scalecultures were grown, lysed, and lyophilized to powder for variantslisted in Table 56.3.

TABLE 56.1 β1, 2GT Round 16 Combinatorial Variants and RebD Levels SEQID NO: Increased (nt/aa) Amino Acid Differences (Relative to SEQ ID NO:4256) RebD^(a) 4497/4498 R427S + 4499/4500 S153A/F155M/R427T + 4501/4502S153A + 4503/4504 F155M/F156W + 4505/4506 K62A/S153A/F155M/F156W/L159V+++ 4507/4508 S153A/F156W/L159V + 4509/4510S153A/F155M/P237L/M238T/T239F/T406Q +++ 4511/4512S153A/F156W/N199S/P237L/R427T ++ 4513/4514 S153A/L159V/P237L/M238G/S352A++ 4515/4516 F156W/N199S/R427T +++ 4517/4518 F156W/N199S + 4519/4520S153A/F156W/R427T +++ 4521/4522 F155M/F156W/R176Q/M181L/N199S ++4523/4524 F155M/F156W/R176Q/M238T/R4275 + 4525/4526K62A/S153A/F155M/N1995/T406Q ++ 4527/4528 S153A/R176Q/M181L/R4275 ++4529/4530 S153A/F155M/M238T/T239F + 4531/4532F155M/M181L/N199S/M238G/T406Q +++ 4533/4534S153A/F155M/F156W/L159V/N199S/M238G/T406Q + 4535/4536K62A/S153A/F155M/F156W/L159V/R427T + 4537/4538 S153A/F155M/F156W ++4539/4540 H164E/I375L/N433D + 4541/4542 I375L ++ 4543/4544I50V/Q137R/P189G/I375L ++ 4545/4546 T223A/I375L + 4547/4548Q137R/H164E/I375L + 4549/4550 S8V/I375L + ^(a)Levels of increasedproduction were determined relative to the reference polypeptide of SEQID NO: 4256, and defined as follows: “+” = production at least1.75-fold, but less than 2.08-fold; “++” = at least 2.08-fold, but lessthan 2.33-fold; and “+++” = at least 2.33-fold increased production, ascompared to the reference polypeptide.

TABLE 56.2 β1, 2GT Round 16 Saturation Mutagenesis Variants and RebDLevels Amino Acid Differences SEQ ID NO: (Relative to SEQ Increased(nt/aa) ID NO: 4256) RebD^(a) 4551/4552 Y449K +++ 4553/4554 A106S +4555/4556 N445R ++ 4557/4558 E192L +++ 4559/4560 F280W ++ 4561/4562S110G + 4563/4564 L1161 + 4565/4566 N115A + 4567/4568 A106K + 4569/4570Q132R +++ 4571/4572 K416R ++ 4573/4574 Y449Q + 4575/4576 V385S ++4577/4578 S9G + 4579/4580 A106R ++ 4581/4582 T220S + 4583/4584 H99R +4585/4586 T220Q ++ 4587/4588 N445K ++ 4589/4590 N302P + 4591/4592 G395Q+++ 4593/4594 N65F ++ 4595/4596 V385P +++ 4597/4598 E192A + 4599/4600A200S ++ 4601/4602 E131C +++ 4603/4604 R423L +++ 4605/4606 N447L ++4607/4608 E136R ++ 4609/4610 Y449R ++ 4611/4612 N302S + 4613/4614 E131S++ 4615/4616 N115T + 4617/4618 P335R + 4619/4620 E192P + 4621/4622Q399K + 4623/4624 M238L + 4625/4626 K245P ++ 4627/4628 D190T +++4629/4630 Q132T + 4631/4632 T12S + 4633/4634 1412R +++ 4635/4636 E3041 +4637/4638 R272H + 4639/4640 M238I + 4641/4642 A170E +++ 4643/4644 P335K++ 4645/4646 T220M +++ 4647/4648 H68I + 4649/4650 E136S + 4651/4652 G10D++ 4653/4654 N257H +++ 4655/4656 Q178L + 4657/4658 P194F +++ 4659/4660V385A + 4661/4662 Y449L + 4663/4664 V385C + 4665/4666 E136G + 4667/4668I242L ++ 4669/4670 P408E + 4671/4672 E136D + 4673/4674 G7H + 4675/4676N445H + 4677/4678 K402R + 4679/4680 E53N ++ 4681/4682 Q132G +++^(a)Levels of increased production were determined relative to thereference polypeptide of SEQ ID NO: 4256, and defined as follows: “+” =production at least 1.07-fold, but less than 1.27-fold; “++” = at least1.27-fold, but less than 1.42 fold; and “+++” = at least 1.42-foldincreased production, as compared to the reference polypeptide.Shake Flask Powder Characterization Assay and Analysis for GlucosylTransfer from Sucrose to ADP to Rebaudioside A

A shake flask powder loading dose response experiment was performed tocharacterize activity of the engineered round 16 variants onrebaudioside A. Levels of 0.003-0.1 g/L shake flask powder (SFP) werepreincubated at 79° C. for 0.5 hand then added to a 100 μL totalreaction volume containing 50 mM potassium phosphate buffer, pH6, 20 mMrebaudioside A, 0.2 mM ADP, 50 mM sucrose, and 0.05 g/L SUS SFP SEQ IDNO: 2432. The reaction was performed at 60° C. in a Thermotron®titre-plate shaker at 300 RPM for 4 h. The reactions were diluted,quenched, and analyzed as described above. The production levels ofrebaudioside D by the round 16 variants at 0.0125 g/L SFP loading areshown in Table 56.3. SEQ ID NO: 4550 was the most improved and wasselected as the best enzyme for the catalysis of glycosyltransfer fromADP-glucose to rebaudioside A to form rebaudioside D.

TABLE 56.3 β1, 2GT Round 16 Shake Flask Variants and RebD Levels SEQ IDNO: Amino Acid Differences Increased (nt/aa) (Relative to SEQ ID NO:4256) RebD^(a) 4549/4550 S8V/I375L +++ 4539/4540 H164E/1375L/N433D +++4497/4498 R427S ++ 4545/4546 T223A/I375L ++ 4515/4516F156W/N199S/R427T + 4531/4532 F155M/M181L/N199S/M238G/T406Q + 4543/4544I50V/Q137R/P189G/I375L + 4501/4502 S153A + ^(a)Levels of increasedproduction were determined relative to the reference polypeptide of SEQID NO: 4256, and defined as follows: “+” = production at least that ofthe reference polypeptide, but less than 1.3-fold; “++” = at least1.3-fold, but less than 1.6-fold; and “+++” = at least 1.6-foldincreased production, as compared to the reference polypeptide.

Example 57 Beta-1,2-ADP-Glycosyltransferase Variants of SEQ ID NO: 4550

In this Example, experiments for evolution and screening of GTpolypeptides derived from SEQ ID NO: 4550 for improved glucosylation ofsteviol glycosides using ADP-glucose are described. Directed evolutionof the GT encoded by SEQ ID NO: 4549 was carried out by constructingcombinatorial libraries of variant genes. Libraries recombined mutationsassociated with improved production identified during the development ofthe present invention. These libraries were then plated, grown, andscreened using the HTP assay described below to provide a seventeenthround (“Round 17”) of engineered GT variant polypeptides withglucosyltransferase activity toward ADP-glucose and steviol glycosides.Seventy-three engineered variants were identified from the recombinedbeneficial mutations (Table 57.1).

HTP Coupled Assay for Glucose Transfer from Sucrose to ADP toRebaudioside A60

Assays were performed on 96-well plates of cleared E. coli culturelysates expressing SEQ ID NO: 4549 variants. Lysis buffer volume was 400μL, and the lysate was diluted 50 or 100-fold into 20 g/L rebaudiosideA60% and pre-incubated for 2 h at 75′C. Assays were then conducted with10 μL diluted lysate, 0.1 g/L SUS SFP SEQ ID NO: 2510, and 0.2 g/Lβ-1,3-glycosyltransferase (β1,3GT) SFP SEQ ID NO: 6864 in 100 μLreaction volume with substrate loading of 20 g/L rebaudioside A 60%(RebA60) and co-substrate loadings of 0.1 g/L ADP (Amresco, ultra puregrade) and 40 g/L sucrose. The following reaction conditions were used:50 mM potassium phosphate buffer, pH6, 60° C. in a Thermotron®titre-plate shaker with 300 RPM shaking for 16-18 h. The reactions weresolubilized by 40× dilution into water, quenched by 5× dilution intoacetonitrile with 0.2% formic acid, precipitated by centrifugation, anddiluted 10× into water for analysis as described above. The resultingengineered variants with glucosyltransferase activity coupled with a SuSon rebaudioside A are listed in Table 57.1. Shake-flask scale cultureswere grown, lysed, and lyophilized to powder for variants listed inTable 57.2.

TABLE 57.1 β1, 2GT Round 17 Combinatorial Variants and RebM Levels SEQID NO: Increased (nt/aa) Amino Acid Differences (Relative to SEQ ID NO:4550) RebM^(a) 7215/7216 R427S ++ 7217/7218 N65G/P143K/Q235R/R427S +7219/7220 N65G/Q1141/K422L/R427S + 7221/7222 R427S/E429D ++ 7223/7224R418A/R427S + 7225/7226 N65G/Q1141/R427S ++ 7227/7228 N65G/P143K +7229/7230 Y44H/A73H/Q1271/L135E ++ 7231/7232Q11L/A73S/Q132K/L135E/P408A + 7233/7234 Q11L/F41H/Q132K/V278L +++7235/7236 Q11L/Q1271/Q132K/L135E/T406S +++ 7237/7238 Q132K/L135E/R1821++ 7239/7240 L45Y/T406G ++ 7241/7242 R182L + 7243/7244L45Y/A73H/Q132K/L135E/R182L +++ 7245/7246L45Y/A73H/Q1271/Q132K/L135E/T406G/P408A +++ 7247/7248Q11L/F41H/Y44H/Q1271/Q132K/T406G + 7249/7250 Q11L/R182L/T406S +7251/7252 L45Y/Q1271/Q132K/L135E/R182T/T406G/P408A + 7253/7254Q11L/A73H/Q1271/Q132K/T406G/P408A +++ 7255/7256 A73H/Q1271 ++ 7257/7258Y44H/L45Y/Q1271/Q132K/L135E/G138T/R182L/T406S/P408A + 7259/7260Q11L/F41H/L45Y/A73H/Q1271/V278L/T406G/P408A + 7261/7262Q11L/Q1271/Q132K/L135E +++ 7263/7264Q11L/L45Y/Q1271/Q132K/L135E/G138T/R182T ++ 7265/7266Y44H/L45Y/Q127I/Q132K/V278L +++ 7267/7268Y44H/A73S/Q132K/L135E/T406G/P408A ++ 7269/7270 Q132K/L135E/T406G +7271/7272 F41H/A73H/Q132K/L135E/T406G + 7273/7274Q11L/Y44H/A73S/Q1271/Q132K/L135E/T406G/P408A + 7275/7276Q11L/Y44H/Q1271 + 7277/7278 Q11L/Y44H/Q132K/V278L/T406G + 7279/7280Q11L/Y44H/A73H/Q1271/Q132K/L135E ++ 7281/7282 Q11L/Q1271/Q132K/R182T +7283/7284 Q11L/A73H/Q1271/Q132K/V278L/T406G/P408A +++ 7285/7286F41H/Y44H/L45Y/A73H/Q1271/Q132K/V278L +++ 7287/7288F41H/L45Y/Q1271/Q132K/L135E/V278L/T406S +++ 7289/7290 L45Y/A73H/Q132K+++ 7291/7292 Q11L/L45Y/T406G ++ 7293/7294 Q11L/A73H/Q132K ++ 7295/7296L45Y/Q1271/Q132K +++ 7297/7298 T406G + 7299/7300 L45Y/Q132K/P408A ++7301/7302 A73H/Q1271/Q132K ++ 7303/7304 Y44H/L45Y/A73H/Q132K/L135E/T406G++ 7305/7306 Q11L/Q1271/Q132K/G138T/R182L +++ 7307/7308Q11L/A73S/Q1271/Q132K/L135E/R182L/V278L +++ 7309/7310Q11L/F41H/L45Y/Q1271/V278L/T406G ++ 7311/7312L45Y/Q1271/Q132K/L135E/R182L ++ 7313/7314Y44H/A73S/Q1271/L135E/R182L/V278L/T406G/P408A + 7315/7316 Q1271 ++7317/7318 Q132K/L135E ++ 7319/7320 L45Y/Q127I/L135E ++ 7321/7322Q1271/Q132K/L135E/R1821/T406G + 7323/7324 Q132K/T406S ++ 7325/7326Q11L/L45Y/A73H/Q1271/L135E + 7327/7328 Q11L/F41H/Y44H/Q1271N278L/T406S +7329/7330 L45Y/A73S/V278L/T406G/P408A ++ 7331/7332Q1271/Q132K/L135E/R182L + 7333/7334 Q11L/Y44H/L45Y/Q1271 + 7335/7336Q11L/Q1271/Q132K/L135E/R182L + 7337/7338 Q11L/Q132K/L135E/T406G/ ++7339/7340 H164E/T220G/K309S/Y449H + 7341/7342 H164E/T220G/Y449L +7343/7344 K309P/Y449L + 7345/7346 K57G + 7347/7348 H164E + 7349/7350Y449L + 7351/7352 K309R + 7353/7354 T220G/Y449H + 7355/7356K56R/K309P/Y449L + 7357/7358 H164E/Y449H + 7359/7360 K309T/Y449H +^(a)Levels of increased production were determined relative to thereference polypeptide of SEQ ID NO: 4550, and defined as follows: “+” =production at least 1.2-fold, but less than 1.52-fold; “++” = at least1.52-fold, but less than 2.1-fold; and “+++” = at least 2.1-foldincreased production, as compared to the reference polypeptide.Shake Flask Powder Characterization Assay and Analysis for GlucosylTransfer from Sucrose to ADP to Rebaudioside A60

A shake flask powder loading dose response experiment was performed tocharacterize activity of the engineered round 17 variants onrebaudioside A60%. Levels of 0.0003-0.04 g/L shake flask powder (SFP)were assayed in 100 μL total reaction volume containing 50 mM potassiumphosphate buffer, pH6, 20 g/L RebA60, 0.1 g/L ADP, 40 g/L sucrose, 0.1g/L SUS SFP SEQ ID NO: 2510, and 0.2 g/L β-1,3-glycosyltransferase(β1,3GT) SFP SEQ ID NO: 6864. The reaction was performed at 60° C. in aThermotron® titre-plate shaker at 300 RPM for 16-18 h. The reactionswere diluted, quenched, and analyzed as described above. The results forthe production of rebaudioside M in the one-pot reaction by the round 17variants at 0.005 g/L SFP loading are shown in Table 57.2. SEQ ID NO:7324 was identified as being the most improved and was selected as thebest enzyme for the catalysis of glycosyltransfer from ADP-glucose torebaudioside A to form rebaudioside D.

TABLE 57.2 β1, 2GT Round 17 Shake Flask Variants and RebM Levels SEQ IDNO: Increased (nt/aa) Amino Acid Differences (Relative to SEQ ID NO:4550) RebM^(a) 7215/7216 R427S − 7225/7226 N65G/Q114I/R427S − 7227/7228N65G/P143K + 7265/7266 Y44H/L45Y/Q127I/Q132K/V278L − 7273/7274Q11L/Y44H/A73S/Q1271/Q132K/L135E/T406G/P408A − 7285/7286F41H/Y44H/L45Y/A73H/Q1271/Q132K/V278L − 7323/7324 Q132K/T406S ++7343/7344 K309P/Y449L + ^(a)Levels of increased production weredetermined relative to the reference polypeptide of SEQ ID NO: 4550, anddefined as follows: “−” = production less than that of the referencepolypeptide; “+” = production at least that of the referencepolypeptide, but less than 1.15-fold; and “++” = at least 1.15-foldincreased production, as compared to the reference polypeptide.

Example 58 Beta-1,2-ADP-Glycosyltransferase Variants of SEQ ID NO: 7324

In this Example, experiments for evolution and screening of GTpolypeptides derived from SEQ ID NO: 7324 for improved glucosylation ofsteviol glycosides using ADP-glucose are described. Directed evolutionof the GT encoded by SEQ ID NO: 7323 was carried out by constructingcombinatorial libraries of variant genes. Libraries recombined mutationsassociated with improved production identified during the development ofthe present invention and subjected certain structural features of theenzyme with saturation mutagenesis. These libraries were then plated,grown, and screened using the HTP assay described below to provide aneighteenth round (“Round 18”) of engineered GT variant polypeptides withglucosyltransferase activity toward ADP-glucose and steviol glycosides.Ninety engineered variants were identified from the recombinedbeneficial mutations (Table 58.1), and 124 were identified fromsaturation mutagenesis (Table 58.2).

HTP Coupled Assay for Glucose Transfer from Sucrose to ADP toRebaudioside A 60

Assays were performed on 96-well plates of cleared E. coli culturelysates expressing SEQ ID NO: 7323 variants. Lysis buffer volume was 400μL, and the lysate was diluted 80-fold into 20 g/L rebaudioside A 60%and pre-incubated for 1.5 h at 75° C. Assays were then conducted with 10μL diluted lysate, 0.05 g/L SUS SFP SEQ ID NO: 7506, and 0.1 g/Lβ-1,3-glycosyltransferase (β1,3GT) SFP SEQ ID NO: 7388, in 100 μLreaction volume with substrate loading of 20 g/L rebaudioside A 60%(RebA60) and co-substrate loadings of 0.05 g/L ADP (Amresco, ultra puregrade) and 40 g/L sucrose. The following reaction conditions were used:50 mM potassium phosphate buffer, pH 6, 60° C. in a Thermotron®titre-plate shaker with 300 RPM shaking for 16-18 h. The reactions weresolubilized by 40× dilution into water, quenched by 5× dilution intoacetonitrile with 0.2% formic acid, precipitated by centrifugation, anddiluted 10× into water for analysis as described above. The resultingengineered variants with glucosyltransferase activity coupled with a SuSon RebA60 are listed in Table 58.1. For the round 18 saturationmutagenesis library, screening was performed as described previously,with the exception that the lysate was diluted 100-fold into 50 mM KphospH6 and pre-incubated for 1 h at 75° C. The resulting engineered GTvariant polypeptides are listed in Table 58.2. Shake-flask scalecultures were grown, lysed, and lyophilized to powder for variantslisted in Table 58.3.

TABLE 58.1 β1, 2GT Round 18 Combinatorial Variants and RebM Levels SEQID NO: Amino Acid Differences Increased (nt/aa) (Relative to SEQ ID NO:7324) RebM^(a) 7765/7766 L1161/D190T/S406G/P408A ++ 7767/7768L1161/M238L/K245P + 7769/7770 D190T/P194F/1412R + 7771/7772D190T/P194F/P335R/K416R + 7773/7774 D190T/P194F ++ 7775/7776D190T/E192L/P194F +++ 7777/7778 A170E/P194F/P335R/K416R + 7779/7780E192L/P194F + 7781/7782 A170E/P335K/K416R ++ 7783/7784D190T/P194F/M238L/K245P ++ 7785/7786 P194F ++ 7787/7788A170E/E192L/P194F/P335K + 7789/7790 A170E/P194F + 7791/7792A170E/D190T/E192L/P194F ++ 7793/7794 D190T + 7795/7796 N115A/D190T/P194F++ 7797/7798 A170E/M238L + 7799/7800 A170E/E192L + 7801/7802N115T/L1161/V278L + 7803/7804 D190T/K245P/I412R + 7805/7806 A170E/P335K++ 7807/7808 L1161/A170E/D190T ++ 7809/7810 L1161/K416R + 7811/7812N115A/A170E/D190T/M238L/1412R + 7813/7814 N115A/K245P/V278L +++7815/7816 E192L/P194F/1242L/S406G/P408A +++ 7817/7818 L1161/M238L +7819/7820 A170E/E192L/P194F/V278L ++ 7821/7822 A170E ++ 7823/7824 V278L++ 7825/7826 R272H/V385P ++ 7827/7828 G10D/Q1271/K132R/V385P + 7829/7830G10D/A73H + 7831/7832 G10D/E53N/R423L ++ 7833/7834E53N/K132G/E136R/N302P/R423L/R427S + 7835/7836 G10D/K132R/N302P/V385P+++ 7837/7838 G10D + 7839/7840 K132G + 7841/7842 A73H/V385S/R427S +++7843/7844 A73H +++ 7845/7846 V385S/R427S + 7847/7848 N302P/V385S +7849/7850 G10D/E53N/R272H/R423L/R427S + 7851/7852 Q1271 + 7853/7854E53N/Q1271/K132R/V385S ++ 7855/7856 N257H/V385S ++ 7857/7858 N302P/V385P+++ 7859/7860 E53N/A73H/A200S/R423L ++ 7861/7862A73H/K132R/E136R/V385S/R427S ++ 7863/7864 V385S +++ 7865/7866G10D/A73H/Q127I +++ 7867/7868 G7V/G10D/A73H/Q1271/K132G/V385P/R423L ++7869/7870 R423L/R427S +++ 7871/7872 A200S +++ 7873/7874 A73H/V385S +++7875/7876 E53N/Q1271/E136R/V385L +++ 7877/7878 R272H/N302P +++ 7879/7880E53N/K132G/N302P/V385S/R423L/R427S +++ 7881/7882 A200S/V385S +++7883/7884 G10D/E53N/A73H/H99R ++ 7885/7886 A73H/Q1271/R427S +++7887/7888 T220M/N445K + 7889/7890 N65G/T220Q/N445R/Y449K + 7891/7892Y449L + 7893/7894 Q399K/S406G + 7895/7896 Q399K/S 406G/Y449L +++7897/7898 P143K/K309P/N445K/N447L/Y449L + 7899/7900 N65G/K402R/S406G/N445R/Y449R ++ 7901/7902 P143K/K309P/K402R + 7903/7904 N65G/Q399K/S406G/N447L/Y449R + 7905/7906 T220Q/N445K/N447L + 7907/7908 N445K/Y449L +7909/7910 K309P/Q399K/Y449L ++ 7911/7912 N445R/Y449L + 7913/7914S406G/N445R/Y449K + 7915/7916 N445R/N447L/Y449L ++ 7917/7918N65G/N445K/N447L/Y449R ++ 7919/7920 S406G/N445R/N447L/Y449L ++ 7921/7922S406G/N445K/Y449R +++ 7923/7924 N65G/A106R/N445K/N447L/Y449L ++7925/7926 P143K/T220Q/N445K/N447L + 7927/7928 K402R/N445R/Y449L +7929/7930 N65G/T220Q/K309P/N445R/N447L + 7931/7932A106R/T220Q/Q399K/K402R/S406G + 7933/7934 N65G/N447L/Y449L ++ 7935/7936N447L ++ 7937/7938 K309P/N445K/N447L/Y449L + 7939/7940N65G/T220Q/N445R/Y449R + 7941/7942 T220Q/Q399K + 7943/7944N65G/P143K/T220Q + ^(a)Levels of increased production were determinedrelative to the reference polypeptide of SEQ ID NO: 7324, and defined asfollows: “+” = production at least that of the reference polypeptide,but less than 1.16-fold; “++” = at least 1.16-fold, but less than1.23-fold; and “+++” = at least 1.23-fold increased production, ascompared to the reference polypeptide.

TABLE 58.2 β1, 2GT Round 18 Saturation Mutagenesis Variants and RebMLevels Amino Acid Differences SEQ ID NO: (Relative to SEQ ID Increased(nt/aa) NO: 7324) RebM^(a) 8091/8092 N51Q + 8093/8094 A434C + 8095/8096S64R ++ 8097/8098 V46Q + 8099/8100 V46L ++ 8101/8102 Q11L/Y44H +++8103/8104 V46C + 8105/8106 V46T ++ 8107/8108 A49K ++ 8109/8110D119T/R172H ++ 8I11/8112 V376N + 8113/8114 G41OR + 8115/8116 A130S +8117/8118 A49N/S406G/P408A + 8119/8120 A398R +++ 8121/8122 G356T ++8123/8124 I420F ++ 8125/8126 D274Q + 8127/8128 Q11L/E71P +++ 8129/8130A381C ++ 8131/8132 P118S ++ 8133/8134 D274T +++ 8135/8136 S64G +++8137/8138 A398H ++ 8139/8140 V133H ++ 8141/8142 D119G ++ 8143/8144 P118A++ 8145/8146 A49G ++ 8147/8148 K100R + 8149/8150 G338S + 8151/8152N157E + 8153/8154 V259T + 8155/8156 P118L ++ 8157/8158 A141Q ++8159/8160 D1791 ++ 8161/8162 I43T +++ 8163/8164 S406G/P408A +++8165/8166 P169E +++ 8167/8168 Q441L ++ 8169/8170 Q11L/V287S +++8171/8172 N108G/R172H +++ 8173/8174 V376S +++ 8175/8176 G356S +8177/8178 E71T +++ 8179/8180 1397L/S406G/P408A ++ 8181/8182 I420L +8183/8184 Q11L/E71T + 8185/8186 N51M + 8187/8188 D119S + 8189/8190A398K + 8191/8192 N333S/S406G/P408A + 8193/8194 V287L + 8195/8196A398L + 8197/8198 E437G + 8199/8200 R172H/I420L +++ 8201/8202R172H/E437D + 8203/8204 V42A +++ 8205/8206 V287A ++ 8207/8208 A141G +++8209/8210 S47N +++ 8211/8212 P118A/S406G/P408A + 8213/8214 Q11L/Q441S +8215/8216 P118V +++ 8217/8218 S64L +++ 8219/8220 V417C +++ 8221/8222M275L +++ 8223/8224 V376G +++ 8225/8226 E437V +++ 8227/8228 E396A +++8229/8230 E76H +++ 8231/8232 V133R ++ 8233/8234 T97V + 8235/8236 H2T ++8237/8238 I112T + 8239/8240 I112N + 8241/8242 N157T + 8243/8244 F109V ++8245/8246 S64A + 8247/8248 F109L + 8249/8250 L401T + 8251/8252 V133S +8253/8254 V287S + 8255/8256 D119A + 8257/8258 V287K + 8259/8260 P1181 +8261/8262 I43L + 8263/8264 R394H/I420W + 8265/8266 N108T + 8267/8268V376R + 8269/8270 N288P + 8271/8272 T48S/A398V + 8273/8274 V385A +8275/8276 I112Q + 8277/8278 M181H ++ 8279/8280 V133L + 8281/8282 A134S++ 8283/8284 N157C + 8285/8286 N51V + 8287/8288 N333S/A398T + 8289/8290M181T + 8291/8292 T97L ++ 8293/8294 E396S ++ 8295/8296 H4F + 8297/8298A141S ++ 8299/8300 I112V +++ 8301/8302 V42D/G138C ++ 8303/8304 S64V +8305/8306 E76L + 8307/8308 K117R/N157T/Q301R + 8309/8310 V376M +8311/8312 S64Q + 8313/8314 V417S ++ 8315/8316 V42A/A141P ++ 8317/8318A398M + 8319/8320 A37G + 8321/8322 I112R ++ 8323/8324 I43M ++ 8325/8326F109Y ++ 8327/8328 V357T + 8329/8330 D179R ++ 8331/8332 I43A + 8333/8334G40Q + 8335/8336 V42G +++ 8337/8338 S64F +++ aLevels of increasedproduction were determined relative to the reference polypeptide of SEQID NO: 7324, and defined as follows: “+” = production at least 1.1-fold,but less than 1.22-fold; “++” = at least 1.22-fold, but less than1.3-fold; and “+++” = at least 1.3-fold increased production, ascompared to the reference polypeptide.Shake Flask Powder Characterization Assay and Analysis for GlucosylTransfer from Sucrose to ADP to Rebaudioside A 60

A shake flask powder loading dose response experiment was performed tocharacterize activity of the engineered round 18 variants onrebaudioside A60%. Levels of 0.0013-0.04 g/L shake flask powder (SFP)were assayed in 100 μL total reaction volume containing 50 mM potassiumphosphate buffer, pH6, 20 g/L RebA60, 0.05 g/L ADP, 40 g/L sucrose, 0.05g/L SUS SFP SEQ ID NO: 7506, and 0.15 g/L β-1,3-glycosyltranferase(β1,3GT) SFP SEQ ID NO:7388. The reaction was performed at 60° C. in aThermotron® titre-plate shaker at 300 RPM for 16-18 h. The reactionswere diluted, quenched, and analyzed as described above. The productionlevels of rebaudioside Min the one-pot reaction by the round 18 variantsat 0.005 g/L SFP loading are shown in Table 58.3. The variant with SEQID NO: 7784 was the most improved variant. Thus, it was selected as thebest enzyme for the catalysis of glycosyltransfer from ADP-glucose torebaudioside A to form rebaudioside D.

TABLE 58.3 β1, 2GT Round 18 Shake Flask Variants and RebM Levels SEQ IDNO: Amino Acid Differences Increased (nt/aa) (Relative to SEQ ID NO:7324) RebM^(a) 7783/7784 D190T/P194F/M238L/K245P ++ 7795/7796N115A/D190T/P194F ++ 7805/7806 A170E/P335K − 7813/7814N115A/K245P/V278L + 7841/7842 A73H/V385S/R427S − 7869/7870 R423L/R427S −7885/7886 A73H/Q1271/R427S − 7895/7896 Q399K/S406G/Y449L + 7903/7904N65G/Q399K/S406G/N447L/Y449R ++ 7933/7934 N65G/N447L/Y449L + ^(a)Levelsof increased production were determined relative to the referencepolypeptide of SEQ ID NO: 7324, and defined as follows: “−” = productionless than that of the reference polypeptide; “+” = production at leastthat of the reference polypeptide, but less than 1.08-fold; and “++” =at least 1.08-fold increased production, as compared to the referencepolypeptide.

Example 59 Beta-1,2-ADP-Glycosyltransferase Variants of SEQ ID NO: 7784

In this Example, experiments for evolution and screening of GTpolypeptides derived from SEQ ID NO: 7784 for improved glucosylation ofsteviol glycosides using ADP-glucose are described. Directed evolutionof the GT encoded by SEQ ID NO: 7783 was carried out by constructinglibraries of variant genes. Libraries recombined mutations associatedwith improved production identified during the development of thepresent invention and subjected certain structural features of theenzyme with saturation mutagenesis. These libraries were then plated,grown, and screened using the HTP assay described below to provide annineteenth round (“Round 19”) of engineered GT variant polypeptides withglucosyltransferase activity toward ADP-glucose and steviol glycosides.Eighty-seven engineered variants were identified from the combinatoriallibraries (Table 59.1), and fifty-eight were identified from thesaturation mutagenesis libraries (Table 59.2).

HTP Coupled Assay for Glucose Transfer from Sucrose to ADP toRebaudioside A 60

Assays were performed on 96-well plates of cleared E. coli culturelysates expressing SEQ ID NO: 7783 variants. Lysis buffer volume was 400μL, and the lysate was diluted 100-fold into 50 mM potassium phosphate,pH 6.0, and pre-incubated for 1 h at 75° C. Assays were then conductedwith 10 L diluted lysate, 0.04 g/L SUS SFP SEQ ID NO: 8420, and 0.1 g/Lβ-1,3-glycosyltransferase (β1,3GT) SFP SEQ ID NO: 8088, in 100 μLreaction volume with substrate loading of 20 g/L rebaudioside A 60%(RebA60) and co-substrate loadings of 0.025 g/L ADP (Amresco, ultra puregrade) and 40 g/L sucrose. The following reaction conditions were used:50 mM potassium phosphate buffer, pH 6, 60° C. in a Thermotron®titre-plate shaker with 300 RPM shaking for 16-18 h. The reactions weresolubilized by 20× dilution into water, quenched by 5× dilution intoacetonitrile with 0.2% formic acid, precipitated by centrifugation, anddiluted 20× into water for analysis as described above. The resultingengineered variants with glucosyltransferase activity coupled with a SuSon RebA60 are listed in Table 59.1 and 59.2. Shake-flask scale cultureswere grown, lysed, and lyophilized to powder for variants listed inTable 59.3.

TABLE 59.1 β1, 2GT Round 19 Combinatorial Variants and RebM Levels SEQID NO: Increased (nt/aa) Amino Acid Differences (Relative to SEQ ID NO:7784) RebMa 8623/8624 S64R + 8625/8626 I112T +++ 8627/8628Q11L/S64R/F109V ++ 8629/8630 S64R/N65G/F109V + 8631/8632 Q11L ++8633/8634 S64R/N65G/I112T ++ 8635/8636 S64R/N65G/I112N ++ 8637/8638F109V/I112T + 8639/8640 F109V/S406G + 8641/8642 F109L/I112T + 8643/8644F109V ++ 8645/8646 F109V/A134S + 8647/8648 I112N/N445K + 8649/8650S64R/N445K ++ 8651/8652 S64R/A134S + 8653/8654 F109V/N115A/P118V +++8655/8656 N65G/I112N/N445K + 8657/8658 S64R/I112N + 8659/8660Q11L/N445K + 8661/8662 N65G/I112N + 8663/8664 V42G/I43M + 8665/8666V46T/N51Q + 8667/8668 A141P + 8669/8670 A73H/A141P +++ 8671/8672 A141G++ 8673/8674 A73H/N302P ++ 8675/8676 E71T/A73H/A141S ++ 8677/8678 E71T++ 8679/8680 E71T/N302P + 8681/8682 A73H ++ 8683/8684 A73H/A141G +++8685/8686 E71T/A141P +++ 8687/8688 A141S/N302P +++ 8689/8690E71T/A73H/A141G ++ 8691/8692 A73H/L116I/A141P + 8693/8694 N302P +8695/8696 A73H/A141S + 8697/8698 A141S + 8699/8700 V46L/S47N/N51Q ++8701/8702 V42A/Y44H/E71T/A73H/L116I ++ 8703/8704 A141G/N302P + 8705/8706S47N/A49G + 8707/8708 S47N/N51V + 8709/8710 E71T/A73H + 8711/8712I43T/A73H/A141P + 8713/8714 V287L/N288P ++ 8715/8716 V287S/A398T +8717/8718 V287S/Q399K +++ 8719/8720 Q1271/P169E/R172H + 8721/8722V376M/A398M + 8723/8724 Q399K +++ 8725/8726 V287S/V376M + 8727/8728A398R ++ 8729/8730 V287M ++ 8731/8732 A398M/Q399K/I420F ++ 8733/8734N288P/Q399K +++ 8735/8736 Q1271/P169E/A398L/Q399K +++ 8737/8738V287L/Q399K +++ 8739/8740 A398M ++ 8741/8742 I420F + 8743/8744A398T/Q399K + 8745/8746 Q1271/V287S + 8747/8748 P169E/A398T ++ 8749/8750P169E/R172H/V287S + 8751/8752 Q1271 + 8753/8754 V376M/Q399K + 8755/8756N288P/A398K + 8757/8758 Q1271/P169E/V287S ++ 8759/8760 Q1271/V376M +++8761/8762 V376M + 8763/8764 Q1271/P169E/V376M/A398M/Q399K + 8765/8766A398M/Q399K ++ 8767/8768 A398L/Q399K + 8769/8770 P169E/R172H/A398M/Q399K++ 8771/8772 V376M/A398L ++ 8773/8774 P169E/R172H/N288P + 8775/8776V287S +++ 8777/8778 L1161/V287S + 8779/8780 N288P +++ 8781/8782P169E/A398M/Q399K ++ 8783/8784 A398R/Q399K ++ 8785/8786N288P/V376S/A398L + 8787/8788 A398L +++ 8789/8790 P169E/V287S +++8791/8792 V287S/I420F/R423L/R427S +++ 8793/8794 A398L/R427S ++ 8795/8796P169E/R172H/N288P/A398L/Q399K/I420F/R423L/R427S + ^(a)Levels ofincreased production were determined relative to the referencepolypeptide of SEQ ID NO: 7784, and defined as follows: “+” = productionat least 1.1-fold, but less than 1.18-fold; “++” = at least 1.18-fold,but less than 1.24-fold; and “+++” = at least 1.24-fold increasedproduction, as compared to the reference polypeptide.

TABLE 59.2 β1,2GT Round 19 Saturation Mutagenesis Variants and RebMLevels SEQ ID NO: Amino Acid Differences Increased (nt/aa) (Relative toSEQ ID NO: 7784) RebM^(a) 9107/9108 Q127A + 9109/9110 L16T ++ 9111/9112I331V ++ 9113/9114 V15L/R394H ++ 9115/9116 Y123A ++ 9117/9118 I202Y +9119/9120 A22G + 9121/9122 P143G ++ 9123/9124 L16A +++ 9125/9126L1161/P143R + 9127/9128 G198Q +++ 9129/9130 F125L +++ 9131/9132 V144S+++ 9133/9134 F156V + 9135/9136 Y268W +++ 9137/9138 Q127G +++ 9139/9140G201K +++ 9141/9142 I331C + 9143/9144 L116I/M350L ++ 9145/9146 L36M +9147/9148 G201A + 9149/9150 L116I ++ 9151/9152 N199S ++ 9153/9154 H93A +9155/9156 Q127V ++ 9157/9158 V144G + 9159/9160 L16G + 9161/9162 P197H +9163/9164 G201N + 9165/9166 H324R + 9167/9168 N199Y + 9169/9170 Y123L+++ 9171/9172 G201L + 9173/9174 Y123V ++ 9175/9176 L13Q + 9177/9178I202A + 9179/9180 G10D/V144L +++ 9181/9182 Y123S +++ 9183/9184 V144Q +9185/9186 G186N ++ 9187/9188 Y123G + 9189/9190 L116I/Y123S ++ 9191/9192G96M + 9193/9194 M350L + 9195/9196 V287A ++ 9197/9198 T187Y + 9199/9200M203T ++ 9201/9202 R14Q + 9203/9204 N199R ++ 9205/9206 G89A ++ 9207/9208VI5A ++ 9209/9210 G10D/N199G +++ 9211/9212 L116I/Y123N + 9213/9214 A149S++ 9215/9216 T187G + 9217/9218 N199G +++ 9219/9220 N199P ++ 9221/9222G10D +++ ^(a)Levels of increased production were determined relative tothe reference polypeptide of SEQ ID NO: 7784, and defined as follows:“+” = production at least 1.1-fold, but less than 1.18-fold; “++” = atleast 1.18-fold, but less than 1.27-fold; and “+++” = at least 1.27-foldincreased production, as compared to the reference polypeptide.Shake Flask Powder Characterization Assay and Analysis for GlucosylTransfer from Sucrose to ADP to Rebaudioside A60

A shake flask powder loading dose response experiment was performed tocharacterize activity of the engineered round 19 variants onrebaudioside 60%. Levels of 0.0013-0.04 g/L shake flask powder (SFP)were assayed in 100 total reaction volume containing 50 mM potassiumphosphate buffer, pH6, 20 g/L RebA60 or RebA97, 0.025 g/L ADP, 20(single substrate) or 40 g/L (one-pot) sucrose, 0.04 g/L SUS SFP SEQ IDNO: 8420, and, for the one-pot reaction only, 0.15 g/Lβ-1,3-glycosyltransferase (β1,3GT) SFP SEQ ID NO: 8088. The reaction wasperformed at 60° C. in a Thermotron® titre-plate shaker at 300 RPM for16-18 h. The reactions were diluted, quenched, and analyzed as describedabove. The production levels of rebaudioside Din the single substrateand rebaudioside Min the one-pot reactions by the round 19 variants at0.0025 and 0.005 g/L SFP loading, respectively, are shown in Table 59.3.The variant with SEQ ID NO: 9180 was the most improved variant. Thus, itwas selected as the best enzyme for the catalysis of glycosyltransferfrom ADP-glucose to rebaudioside A to form rebaudioside D.

TABLE 59.3 β1,2GT Round 19 Shake Flask Variants and RebD and RebM LevelsSEQ ID NO: Amino Acid Differences Increased Increased (nt/aa) (Relativeto SEQ ID NO: 7784) RebD^(a) RebM^(a) 8635/8636 S64R/N65G/I112N − +8651/8652 S64R/A134S ++ ++ 8685/8686 E71T/A141P + + 8765/8766A398M/Q399K + + 8789/8790 P169E/V287S − + 9131/9132 V144S + ++ 9169/9170Y123L − − 9179/9180 G10D/V144L + ++ 9181/9182 Y123S − − ^(a)Levels ofincreased production were determined relative to the referencepolypeptide of SEQ ID NO: 7784, and defined as follows: “−“ = productionless than that of the reference polypeptide; “+” = production at leastthat of the reference polypeptide, but less than 1.1-fold; and “++” = atleast 1.1-fold increased production, as compared to the referencepolypeptide.

Example 60 Beta-1,3-ADP-Glycosyltransferase Variants of SEQ ID NO: 696

In this Example, experiments for evolution and screening ofβ1,3-glycosyltransferase (GT) polypeptides derived from SEQ ID NO: 696for improved glucosylation of steviol glycosides using ADP-glucose aredescribed. Directed evolution of the GT encoded by SEQ ID NO: 695 (i.e.,SEQ ID NO: 696) was carried out by constructing libraries of variantgenes in which mutations associated with improved activity in previousrounds above were recombined and in which certain structural featureswere subjected to saturation mutagenesis. These libraries were thenplated, grown, and screened using the high-throughput (HTP) assaydescribed below to provide an eighth round (“Round 8”) of 65 engineeredGT variant polypeptides with glucosyltransferase activity towardADP-glucose and steviol glycosides.

HTP Assay for Glucose Transfer from Sucrose to ADP and then fromADP-Glucose to Stevioside

Assays were performed on 96-well plates of cleared E. coli culturelysates expressing SEQ ID NO: 695 variants. Pellets were lysed with 400μL lysis buffer with 0.5 mg/mL lysozyme and 0.5 mg/mL PMBS in 25 mMTris-HCl pH 7.5 for 1.5 h and cleared by centrifugation. Assays wereconducted with 10 μL lysate in 100 μL reactions and with 5 mMrebaudioside D substrate, 0.2 mM ADP (Sigma, >93% purity) co-substrate,0.1 g/L SUS SFP SEQ ID NO: 1222, and 15 mM sucrose (cane sugar). Thefollowing reaction conditions were used: 50 mM KPhos buffer, pH 7, 3 mMMgCl₂, 50° C. in a Thermotron® titre-plate shaker with 300 RPM shakingfor 4-5 h. Then, reactions were solubilized by 20× dilution in water,quenched with 5× dilution in acetonitrile with 0.2% formic acid,centrifuged 10 m at 4° C., and supernatants were diluted 5× in water foranalysis. Samples were analyzed by RapidFire-MS/MS as described inExample 31, Table 31.1. Glycosyltransferase variant polypeptides thatproduced rebaudioside M from rebaudioside D with with in situsynthesized ADP-glucose at greater quantities than SEQ ID NO: 696 wereidentified. Most were retested in triplicate on stevioside (95% purity).The engineered polypeptides are listed in Table 60.1. Shake-flask scalecultures were grown for SFP production as described in Example 31 foranalysis of variants shown in Table 60.2 relative to SEQ ID NO: 696.

TABLE 60.1 β1,3GT Round 8 Variants and RebM and RebA Levels SEQ ID NO:Amino Acid Differences Increased Increased (nt/aa) (Relative to SEQ IDNO: 696) RebM^(a) RebA^(a) 4683/4684 T262S/W401L ++ ND 4685/4686I189L/M206K/K208A/V365I ++ ND 4687/4688K185R/K208A/E230S/S252N/S255N/D290E/V365I ++ ND 4689/4690V365I/W401L/V413L/V435Q ++ ND 4691/4692 K208A/V365I/V435Q +++ ND4693/4694 S304P/P322S/V365I/W401L ++ ND 4695/4696 L249N + + 4697/4698L249M + + 4699/4700 Q71R + + 4701/4702 L249S + + 4703/4704 I243L + +4705/4706 K51A + ++ 4707/4708 K200N + + 4709/4710 L249Y + + 4711/4712R308F ++ + 4713/4714 P339D + + 4715/4716 L249E + + 4717/4718 L78F + −4719/4720 L78R + − 4721/4722 L249I ++ ++ 4723/4724 R88K + − 4725/4726L78Q + − 4727/4728 L78I + − 4729/4730 C366A + + 4731/4732 F352Q ++ +4733/4734 L245G + + 4735/4736 L78G +++ − 4737/4738 I25V + + 4739/4740L78K ++ − 4741/4742 L78M + − 4743/4744 S374T + + 4745/4746 L249T + +4747/4748 L78P ++ − 4749/4750 K200S + + 4751/4752 N56L + + 4753/4754R308Y ++ − 4755/4756 H259Y + − 4757/4758 H259S +++ ND 4759/4760 T80L +++ND 4761/4762 C366A ++ ND 4763/4764 P22L + − 4765/4766 I243M ++ +4767/4768 I25L +++ + 4769/4770 L249H ++ − 4771/4772 I243C + + 4773/4774S279G +++ +++ 4775/4776 H259G ++ + 4777/4778 L249P ++ ND 4779/4780I243Y + − 4781/4782 L78P +++ ND 4783/4784 L362T ++ +++ 4785/4786 R88V+++ − 4787/4788 L78E +++ − 4789/4790 H81C + − 4791/4792 L78G +++ ND4793/4794 I243V ++ ND 4795/4796 L157G + − 4797/4798 I243V ++ − 4799/4800R88I +++ − 4801/4802 S364G + + 4803/4804 S282T ++ + 4805/4806 S284T + +4807/4808 V338C +++ ++ 4809/4810 S364G ++ ND 4811/4812 L157Q + −^(a)Levels of increased production were determined relative to thereference polypeptide of SEQ ID NO: 696, and defined as follows: “−“ =production less than that of the reference polypeptide; “+” = productionat least that of the reference polypeptide, but less than 1.24-fold;“++” = at least 1.24-fold, but less than 1.49-fold; and “+++” = at least1.49-fold increased production, as compared to the referencepolypeptide. ND = not determined.SFP Characterization Assay and Analysis for Glucosyl Transfer fromADP-Glucose to Stevioside or Rebaudioside D

Shake flask powders (SFP) were reconstituted to a concentration of 20g/L and diluted to 0.002-5 g/L SFP in 100 μL total reaction volumes of50 mM potassium phosphate buffer, pH 7, with 3 mM MgCl₂, 1 mM stevioside(95% purity) or rebaudioside D, and 1 mM ADP-glucose (Sigma, >93%purity). The reactions were incubated at 50° C. in a Thermotron®titre-plate shaker with 300 RPM shaking for 1 hour. The reactions weresolubilized by 5× dilution with water, quenched by 4× dilution inacetonitrile with 0.2% formic acid, cleared by centrifugation, anddiluted 5× with water for analysis. Glycosylated products were detectedin by SPE-QQQ as described in Example 31, Table 31.1.

TABLE 60.2 β1,3GT Round 8 Shake Flask Powder Variants and RebM and RebALevels SEQ ID NO: Amino Acid Differences Increased Increased (nt/aa)(Relative to SEQ ID NO: 696) RebM^(a) RebA^(a) 4683/4684 T262S/W401L + +4685/4686 I189L/M206K/K208A/V365I + + 4693/4694 S304P/P322S/V365I/W401L− + ^(a)Levels of increased production were determined relative to thereference polypeptide of SEQ ID NO: 696, and defined as follows: “−“ =production less than that of the reference polypeptide; and “+” =production at least that of the reference polypeptide, but less than1.35-fold increased production, as compared reference polypeptide.

In these experiments, all of the variants in Table 60.2 (i.e., SEQ IDNOS: 4684, 4686, and 4694) produced rebaudioside A from steviosideand/or rebaudioside M from rebaudioside D with ADP-glucose at greaterquantities than SEQ ID NO: 696. The variant SEQ ID NO: 4684 was selectedfor further directed evolution.

Example 61 Beta-1,3-ADP-Glycosyltransferase Variants of SEQ ID NO: 4684

In this Example, experiments for evolution and screening ofβ1,3-glycosyltransferase (β1,3GT) polypeptides derived from SEQ ID NO:4684 for improved glucosylation of steviol glycosides using in situsynthesized ADP-glucose are described. Directed evolution of the GTencoded by SEQ ID NO: 4684 (i.e., SEQ ID NO: 4683) was carried out byconstructing a library of variant genes in which mutations associatedwith improved activity in previous rounds above were recombined. Theselibraries were then plated, grown, and screened using thehigh-throughput (HTP) assay described below to provide a ninthround(“Round 9”) of 31 engineered GT variant polypeptides withglucosyltransferase activity toward ADP-glucose and steviol glycosides.

HTP Assay for Glucose Transfer from Sucrose to ADP and then fromADP-Glucose to Stevioside

Assays were performed on 96-well plates of cleared E. coli culturelysates expressing SEQ ID NO: 4684 variants. Pellets were lysed, andlysate was cleared as described in Example 60. Assays were conductedwith 10 μL lysate in 100 μL reactions and with 5 mM stevioside (>95%purity) substrate, 0.2 mM ADP (Sigma, >93% purity) co-substrate, 0.1 g/LSUS SFP SEQ ID NO: 1222, and 15 mM sucrose (cane sugar). The followingreaction conditions were used: 50 mM KPhos buffer, pH 7, 3 mM MgCl₂, 50°C. in a Thermotron® titre-plate shaker with 300 RPM shaking for 4-5 h.Reactions were solubilized, quenched, and diluted as described inExample 60. Samples were analyzed by RapidFire-MS/MS as described inExample 31, Table 31.1. Glycosyltransferase variant polypeptides thatproduced rebaudioside A from stevioside with in situ synthesizedADP-glucose at greater quantities than SEQ ID NO: 4684 were identified.The engineered polypeptides are listed in Table 61.1. Shake-flask scalecultures were grown, lysed, cleared, and lyophilized to powder asdescribed in Example 31 for analysis of variants shown in Table 61.2relative to SEQ ID NO: 4684.

TABLE 61.1 β1,3GT Round 9 Variants and RebA Levels SEQ ID NO: Amino AcidDifferences Increased (nt/aa) (Relative to SEQ ID NO: 4684) RebA^(a)4813/4814 N56H/L249M/S284T/W353Y + 4815/4816K51A/1243L/L249E/R308F/W353Y ++ 4817/4818 K51A/N56H/L249M/L362M/C366S ++4819/4820 K51A/N56H/L249Y/W353Y + 4821/4822 I243L/R308F/W353Y +++4823/4824 K51A/L249Y/S284T/R308F/L362M/C366V +++ 4825/4826K51A/N56H/L249M/L362M/S364G +++ 4827/4828K51A/N56H/I243L/L249E/R308F/L362M/S364G +++ 4829/4830N56H/I243L/S364G/C366V ++ 4831/4832 N56H/L249M/W353Y ++ 4833/4834N56H/L249Y +++ 4835/4836 R308F/C366A ++ 4837/4838N56H/I243L/L249E/S282T/S364G/C366V +++ 4839/4840 K51A/L249M + 4841/4842N56H + 4843/4844 K51A/L249M/S282T/W353Y/C366S ++ 4845/4846K51A/L249E/W353Y/L362M/S364G + 4847/4848 N56H/S284T/C366V + 4849/4850I243L/S282T/W353Y/L362M/S364G + 4851/4852 K51A/W353Y/L362M + 4853/4854K51A/N56H + 4855/4856 I243L/L249E/S282T/S284T/L362M/S364G/C366S ++4857/4858 S282T + 4859/4860 I243L/L249M/R308F/W353Y/C366A + 4861/4862K51A/N56H/I243L/L249E/S282T/W353Y/L362M/C366S + 4863/4864I243L/S282T/L362M/S364G/C366V ++ 4865/4866K51A/L249M/S282T/S284T/S364G + 4867/4868 L249Y/W353Y/L362M/C366S +4869/4870 L362M/C366A + 4871/4872 I243L/L249Y/W353Y/L362M/C366S +4873/4874 K51A/I243L/L249E/A348S/L362M/C366V ++ ^(a)Levels of increasedproduction were determined relative to the reference polypeptide of SEQID NO: 4684, and defined as follows:? “+” = production at least1.2-fold, but less than 1.42-fold; “++” = at least 1.42-fold, but lessthan 1.59-fold; and “+++” = at least 1.59-fold increased production, ascompared to the reference polypeptide.SFP Characterization Assay and Analysis for Glucosyl Transfer fromADP-Glucose to Stevioside or Rebaudioside D

Shake flask powders (SFP) were reconstituted to a concentration of 20g/L and diluted to 0.002-5 g/L SFP in 100 μL total reaction volumes of50 mM potassium phosphate buffer, pH7, with 3 mM MgCl₂, 1 mM stevioside(95% purity) or rebaudioside D, and 1 mM ADP-glucose (Sigma, >93%purity). The reactions were incubated at 50° C. in a Thermotron®titre-plate shaker with 300 RPM shaking for 1 hour. The reactions weresolubilized by 5× dilution with water, quenched by 4× dilution withacetonitrile and 0.2% formic acid, cleared by centrifugation, anddiluted 5× with water for analysis. Glycosylated products were detectedin by SPE-QQQ as described in Example 31, Table 31.1.

TABLE 61.2 β1,3GT Round 9 Shake Flask Powder Variants and RebM and RebALevels SEQ ID NO: Amino Acid Differences Increased Increased (nt/aa)(Relative to SEQ ID NO: 4684) RebA^(a) RebM^(a) 4837/4838N56H;I243L;L249E;S282T;S364G;C366V; ++ +++ 4821/4822I243L;R308F;W353Y; + ++ 4825/4826 K51A;N56H;L249M;L362M;S364G; + ++4823/4824 K51A;L249Y;S284T;R308F;L362M;C366V; + ++ 4833/4834N56H;L249Y; + + ^(a)Levels of increased production were determinedrelative to the reference polypeptide of SEQ ID NO: 4684, and defined asfollows: “+” = production at least that of the reference polypeptide,but less than 1.4-fold; “++” = at least 1.4-fold, but less than 2-fold;and “+++” = at least 2-fold increased production, as compared to thereference polypeptide.

In these experiments, all of the variants in Table 61.2 (i.e., SEQ IDNOS: 4822, 4824, 4826, 4834, and 4838) produced rebaudioside A fromstevioside and rebaudioside M from rebaudioside D with ADP-glucose atgreater quantities than SEQ ID NO: 4684. The variant of SEQ ID NO: 4838had the highest activity on both stevioside and rebaudioside D withADP-glucose as a co-substrate in these experiments. Thus, the encodingpolynucleotide (SEQ ID NO: 4837) was selected for further directedevolution.

Example 62 Beta-1,3-ADP-Glycosyltransferase Variants of SEQ ID NO: 4838

In this Example, experiments for evolution and screening ofβ1,3-glycosyltransferase (β1,3GT) polypeptides derived from SEQ ID NO:4838 for improved glucosylation of steviol glycosides using in situsynthesized ADP-glucose are described. Directed evolution of the GTencoded by SEQ ID NO: 4837 (i.e., SEQ ID NO: 4838) was carried out byconstructing libraries of variant genes in which mutations associatedwith improved production identified during the development of thepresent invention were recombined and in which certain structuralfeatures were subjected to saturation mutagenesis. These libraries werethen plated, grown, and screened using the high-throughput (HTP) assaydescribed below to provide a tenth round (“Round 10”) of 123 engineeredGT variant polypeptides with glucosyltransferase activity towardADP-glucose and steviol glycosides.

HTP Assay for Glucose Transfer from Sucrose to ADP and then fromADP-Glucose to Rebaudioside D

Assays were performed on 96-well plates of cleared E. coli culturelysates expressing SEQ ID NO: 4838 variants. Pellets were lysed, and thelysate was cleared as described in Example 60, and then diluted 4× intopotassium phosphate buffer, pH 6.5. Assays were conducted with 10 μLlysate in 100 μL reactions and with 5 mM stevioside (>95% purity)substrate, 0.2 mM ADP (Sigma, >93% purity) co-substrate, 0.2 g/L SUS SFPSEQ ID NO: 1222, and 15 mM sucrose (cane sugar). The following reactionconditions were used: 50 mM potassium phosphate buffer, pH 6.5, 50° C.in a Thermotron® titre-plate shaker with 300 RPM shaking for 1 h.Reactions were solubilized, quenched, and diluted as described inExample 60. Samples were analyzed by RapidFire-MS/MS as described inExample 1, Table 1.1. Selected variants were retested in a similar assaywith 10× lysate dilution, 4 mM stevioside or rebaudioside D, 0.375 mMADP, 0.5 g/L sucrose synthase SEQ ID NO: 1392, 30 mM sucrose, and 2 hincubation. Glycosyltransferase variant polypeptides that producedrebaudioside M from rebaudioside D with in situ synthesized ADP-glucoseat greater quantities than SEQ ID NO: 4838 were identified. Theengineered polypeptides are listed in Table 62.1. Shake-flask scalecultures were grown, lysed, cleared, and lyophilized to powder asdescribed in Example 1 for analysis of variants shown in Table 62.2relative to SEQ ID NO: 4838.

TABLE 62.1 β1,3GT Round 10 Variants and RebM and RebA Levels SEQ ID NO:Amino Acid Differences Increased Increased (nt/aa) (Relative to SEQ IDNO: 4838) RebA^(a) RebM^(a) 4875/4876 K51A/H56L/L362M +++ +++ 4877/4878Q177S ++ ++ 4879/4880 K51A ++ ++ 4881/4882 H56L/L362M/V366A ++ +++4883/4884 H56L/L243M/E249Y +++ +++ 4885/4886 K208E ++ ++ 4887/4888 A110S++ +++ 4889/4890 K439D ++ + 4891/4892 H56L/L243M/E249M +++ +++ 4893/4894K208I ++ ++ 4895/4896 Q265A ++ +++ 4897/4898 K208W/V320I +++ ++4899/4900 K208L ++ ++ 4901/4902 K200S/L243M/E249Y/H259G ++ +++ 4903/4904K336E ++ +++ 4905/4906 K208T ++ +++ 4907/4908 K222P +++ +++ 4909/4910H259N ++ ++ 4911/4912 K289H ++ +++ 4913/4914 R234T ++ ++ 4915/4916W353Y/L362M − + 4917/4918 L174S + + 4919/4920 H259S ++ ++ 4921/4922I25V/L362M/V366S − + 4923/4924 E226R + + 4925/4926 K171V + + 4927/4928R234E + ++ 4929/4930 R88I − +++ 4931/4932 L256A + + 4933/4934 E175N + ++4935/4936 V428K + + 4937/4938 E226T + + 4939/4940 K439Q ++ + 4941/4942E405Q + + 4943/4944 R88C/R173S − +++ 4945/4946 I198S − +++ 4947/4948K208G + + 4949/4950 L163K + + 4951/4952 I198D/F313S − + 4953/4954R234A + + 4955/4956 K171D + + 4957/4958 Q177P + + 4959/4960 I198E − +++4961/4962 A85E − ++ 4963/4964 Q209E/R234Q + + 4965/4966K51A/H56L/K200S/L243M/E249I/H259G/V338C ++ +++ 4967/4968 K208N + ++4969/4970 I25L/H56L/W353Y + ++ 4971/4972 I198D/F313S/V428S − ++4973/4974 R411T + +++ 4975/4976 R234H/N408D + + 4977/4978 I198D − +++4979/4980 P322V + + 4981/4982 Q177K + + 4983/4984 Q209A + + 4985/4986K51A/Q71R/E249M/S279G/S284T/L362M/V366S + +++ 4987/4988 K171E + ++4989/4990 K289T + + 4991/4992 K171P + + 4993/4994L243M/V338C/L362M/V366S + ++ 4995/4996 P272D + + 4997/4998 Q159N + ++4999/5000 H259T + + 5001/5002 P70S/I198D/H259E/F313S − ++ 5003/5004I25L + ++ 5005/5006 I25V/L243M/E249Y/H259G/V366A + +++ 5007/5008S253A + + 5009/5010 I25V/L243M/E249I/L362M + +++ 5011/5012 L174E + ++5013/5014 K208V + + 5015/5016 I198T − + ^(a)Levels of increasedproduction were determined relative to the reference polypeptide of SEQID NO: 4838, and defined as follows: “−“ = production less than that ofthe reference polypeptide; “+” = production at least that of thereference polypeptide, but less than 1.34-fold; “++” = at least1.34-fold, but less than 1.54-fold; and “+++” = at least 1.54-foldincreased production, as compared to the reference polypeptide.SFP Characterization Assay and Analysis for Glucosyl Transfer fromADP-Glucose to Stevioside or Rebaudioside D

Shake flask powders (SFP) were reconstituted to a concentration of 20g/L and diluted to 0.006-0.2 g/L SFP in 100 μL total reaction volumes of50 mM potassium phosphate buffer, pH 6.5, 1 mM stevioside (95% purity)or rebaudioside D, and 1 mM ADP-glucose (Sigma, >93% purity). Thereactions were incubated at 50° C. in a Thermotron® titre-plate shakerwith 300 RPM shaking for 1 hour. The reactions were solubilized by 5×dilution with water, quenched by 4× dilution with acetonitrile and 0.2%formic acid, cleared by centrifugation, and diluted 5× with water foranalysis. Glycosylated products were detected in by SPE-QQQ as describedin Example 31, Table 31.1.

TABLE 62.2 β1,3GT Round 10 Shake Flask Powder Variants and RebA and RebMLevels SEQ ID NO: Amino Acid Differences Increased Increased (nt/aa)(Relative to SEQ ID NO: 4838) RebA^(a) RebM^(a) 4875/4876K51A/H56L/L362M + + 4879/4880 L243M/V338C/L362M/V366S − + 4881/4882K200S/L243M/E249Y/H259G − − 4909/4910 I198D/F313S − ++ 4911/4912P70S/I198D/H259E/F313S − − ^(a)Levels of increased production weredetermined relative to the reference polypeptide of SEQ ID NO: 4838, anddefined as follows: “−“ = production less than that of the referencepolypeptide; and “+” = production at least that of the referencepolypeptide, but less than 1.3-fold increased production, as compared tothe reference polypeptide.

In these experiments, one variant in Table 62.2 (i.e., SEQ ID NO: 4876)produced rebaudioside A from stevioside and rebaudioside M fromrebaudioside D with ADP-glucose at greater quantities than SEQ ID NO:4838. Thus, the encoding polynucleotide (SEQ ID NO: 4875) was selectedfor further directed evolution.

Example 63 Beta-1,3-ADP-Glycosyltransferase Variants of SEQ ID NO: 4876

In this Example, experiments for evolution and screening ofβ1,3-glycosyltransferase (β1,3GT) polypeptides derived from SEQ ID NO:4876 for improved glucosylation of steviol glycosides using in situsynthesized ADP-glucose are described. Directed evolution of the GTencoded by SEQ ID NO: 4875 (i.e., SEQ ID NO: 4876) was carried out byconstructing libraries of variant genes in which mutations associatedwith improved production identified during the development of thepresent invention were recombined and in which certain structuralfeatures were subjected to saturation mutagenesis. These libraries werethen plated, grown, and screened using the high-throughput (HTP) assaydescribed below to provide an eleventh round (“Round 11”) of 122engineered GT variant polypeptides with glucosyltransferase activitytoward ADP-glucose and steviol glycosides.

HTP Assay for Glucose Transfer from Sucrose to ADP and then fromADP-Glucose to Stevioside and Rebaudioside D

Assays were performed on 96-well plates of cleared E. coli culturelysates expressing SEQ ID NO: 4875 variants. Pellets were lysed, andlysate was cleared as described in Example 60, and then diluted 10× intopotassium phosphate buffer, pH 6.5. Assays of combinatorial librarieswere conducted with 10 μL lysate in 100 μL reactions and with 4 mMstevioside (>95% purity) or rebaudioside D substrate, 0.2 mM ADP(Sigma, >93% purity) co-substrate, 0.2 g/L SUS SFP SEQ ID NO: 1456, and24 mM sucrose (cane sugar). The following reaction conditions were used:50 mM potassium phosphate buffer, pH 6.5, 50° C. in a Thermotron®titre-plate shaker with 300 RPM shaking for 1 h. Reactions weresolubilized, quenched, and diluted as described in Example 60. Sampleswere analyzed by RapidFire-MS/MS as described in Example 1, Table 1.1.For the saturation mutagenesis library, the same assay was performed butwith 5× diluted lysate, pH 6 potassium phosphate buffer, and 1-2incubation at 55° C. Selected variants were also retested in triplicateunder these conditions. Glycosyltransferase variant polypeptides thatproduced rebaudioside A/rebaudioside M from stevioside/rebaudioside Dwith in situ synthesized ADP-glucose at greater quantities than SEQ IDNO: 4876 were identified. The engineered polypeptides are listed inTable 63.1. Shake-flask scale cultures were grown, lysed, cleared, andlyophilized to powder as described in Example 1 for analysis of variantsshown in Table 63.2 relative to SEQ ID NO: 4876.

TABLE 63.1 β1,3GT Round 11 Variants and RebM and RebA Levels SEQ ID NO:Amino Acid Differences Increased Increased (nt/aa) (Relative to SEQ IDNO: 4876) RebM^(a) RebA^(a) 5017/5018 I25L/Q209E/L243M/H259G + ++5019/5020 Q209E/E249M/V338C ++ ++ 5021/5022I198D/K200S/Q209E/L243M/E249Y/K289T ++ − 5023/5024 I25L/E2491 ++ ++5025/5026 I25L/Q209E/E249I/H259G/V366A ++ ++ 5027/5028 K200S/Q209E/V366A++ + 5029/5030 I25L/Q209E/H259G ++ ++ 5031/5032 I25L/H259G/S279G/K289T++ ++ 5033/5034 I25L/S279G/S284T/K289T + ++ 5035/5036 I198D ++ −5037/5038 I25L/Q209E/S279G/V366A + ++ 5039/5040 S279G ++ ++ 5041/5042I25L/H259G/S279G ++ ++ 5043/5044 Q209E/E249Y/S279G/S284T/K289T + ++5045/5046 I25L/Q209E/K289T/V366A ++ ++ 5047/5048 V366A + ++ 5049/5050E249M/H259G + ++ 5051/5052 Q209E/E249I/H259G/K289T ++ ++ 5053/5054 K289T++ ++ 5055/5056 Q209E/S279G/K289T ++ ++ 5057/5058 I25L/Q209E/L243M/E249Y++ ++ 5059/5060 I25L/K200S/L243M/E249M ++ + 5061/5062 I25L ++ +5063/5064 Q209E ++ ++ 5065/5066 Q209E/E249Y/H259G/S279G/V338C/V366A ++++++ 5067/5068 I25L/K200S/Q209E/V338C ++ ++ 5069/5070 H259G/V366A ++ +5071/5072 I25H/Q209E/L243M/E249Y ++ + 5073/5074 Q209E/V366A +++ ++5075/5076 I25L/I198D/Q209E +++ − 5077/5078 V338C ++ ++ 5079/5080I25L/K289T ++ ++ 5081/5082 I25L/S279G/S284T ++ ++ 5083/5084 S279G/V338C++ ++ 5085/5086 I198D/E249M/V338C ++ − 5087/5088 I25L/Q209E/L243M ++ ++5089/5090 K53R ++ + 5091/5092 R308T + ND 5093/5094 G364S + ++ 5095/5096R308C + ND 5097/5098 T54P + ND 5099/5100 R308L ++ ++ 5101/5102 S55W + ND5103/5104 K53C + + 5105/5106 K53L + ND 5107/5108 K336Y + ND 5109/5110K247L ++ ++ 5111/5112 K53V ++ ++ 5113/5114 Q341V + ND 5115/5116 K247A++ + 5117/5118 S55T ++ ++ 5119/5120 E342R + ND 5121/5122 K336M + +5123/5124 T54V ++ ++ 5125/5126 L107A ++ + 5127/5128 S252Q + + 5129/5130S254G + + 5131/5132 A426T + ND 5133/5134 R411S + ND 5135/5136 S252E ++++ 5137/5138 K392H ++ ++ 5139/5140 S194Q + ND 5141/5142 V34I + ND5143/5144 L107T ++ + 5145/5146 S253G ++ + 5147/5148 L107C + + 5149/5150P322M + ND 5151/5152 K392Q + + 5153/5154 G297A + ND 5155/5156 F327L ++ +5157/5158 T61S ++ ++ 5159/5160 L174M + ND 5161/5162 T8D + ND 5163/5164R411E + ND 5165/5166 M414L + ND 5167/5168 R412T + ND 5169/5170 S388V +ND 5171/5172 P322T ++ + 5173/5174 S111G + + 5175/5176 S446F + ND5177/5178 K432T + ND 5179/5180 S253P + + 5181/5182 L141M + ND 5183/5184K430R + ND 5185/5186 V9G + ND 5187/5188 S254M ++ ++ 5189/5190 S252A + ND5191/5192 P322R ++ ND 5193/5194 A238K + ND 5195/5196 E321D ++ ND5197/5198 L256D + + 5199/5200 L256T + ND 5201/5202 M108C + ND 5203/5204S111C + ND 5205/5206 L256W + ND 5207/5208 R234H/G297A + ND 5209/5210S254P + + 5211/5212 S449L + + 5213/5214 T8L + ND 5215/5216 R412H + ND5217/5218 S388T + + 5219/5220 D69S + + 5221/5222 P79S + − 5223/5224E201T + + 5225/5226 I91R + + 5227/5228 P70K + − 5229/5230 D69T ++ +5231/5232 W87R ++ − 5233/5234 I91T + + 5235/5236 P79V + − 5237/5238 D69N++ + 5239/5240 P158Q + + 5241/5242 I91Q + − 5243/5244 W87M ++ −5245/5246 E73S ++ + 5247/5248 K190R ++ ++ 5249/5250 A153S ++ ++5251/5252 E201P + ++ 5253/5254 P79G + − 5255/5256 W87L ++ − 5257/5258D69Q + + 5259/5260 K205P − ++ ^(a)Levels of increased production weredetermined relative to the reference polypeptide of SEQ ID NO: 4876, anddefined as follows: “−“ = production less than that of the referencepolypeptide; “+” = production at least that of the referencepolypeptide, but less than 1.2-fold; “++” = at least 1.2-fold, but lessthan 2-fold; and “+++” = at least 2-fold increased production, ascompared to the reference polypeptide. ND = not determined.SFP Characterization Assay and Analysis for Glucosyl Transfer fromSucrose to ADP and then from ADP-Glucose to Stevioside and RebaudiosideD

Shake flask powders (SFP) were reconstituted to a concentration of 20g/L and diluted to 0.006-0.2 g/L SFP in 100 μL total reaction volumes of50 mM potassium phosphate buffer, pH 6.5, 4 mM stevioside (95% purity)or rebaudioside D, 0.2 mM ADP (Sigma, >93% purity) co-substrate, 0.2 g/LSUS SFP SEQ ID NO: 1456, and 24 mM sucrose (cane sugar). The reactionswere incubated at 50° C. in a Thermotron® titre-plate shaker with 300RPM shaking for 1 hour. The reactions were solubilized by 20× dilutionwith water, quenched by 5× dilution in acetonitrile with 0.2% formicacid, cleared by centrifugation, and diluted 5× with water for analysis.Glycosylated products were detected in by SPE-QQQ as described inExample 31, Table 31.1.

TABLE 63.2 β1,3GT Round 11 Shake Flask Powder Variants and RebA and RebMLevels SEQ ID NO: Amino Acid Differences Increased Increased (nt/aa)(Relative to SEQ ID NO: 4876) RebA^(a) RebM^(a) 5075/5076I25L/I198D/Q209E − +++ 5041/5042 I25L/H259G/S279G ++ +++ 5025/5026I25L/Q209E/E249I/H259G/V366A + ++ 5065/5066Q209E/E249Y/H259G/S279G/V338C/V366A ++ ++ 5073/5074 Q209E/V366A + ++5043/5044 Q209E/E249Y/S279G/S284T/K289T + + ^(a)Levels of increasedproduction were determined relative to the reference polypeptide of SEQID NO: 4876, and defined as follows: “−“ = production less than that ofthe reference polypeptide; “+” = production at least that of thereference polypeptide, but less than 1.3-fold; “++” = at least 1.3-fold,but less than 1.8-fold; and “+++” = at least 1.8-fold increasedproduction, as compared to the reference polypeptide.

In these experiments, the six variants in Table 63.2 (i.e., SEQ ID NO:5076, 5042, 5026, 5066, 5074, and 5044) produced rebaudioside A fromstevioside and/or rebaudioside M from rebaudioside D with ADP-glucose atgreater quantities than SEQ ID NO: 4876. SEQ ID NO: 5066 performed bestin the primary screen (Table 63.1) and had the most beneficialmutations. Thus, the encoding polynucleotide (SEQ ID NO: 5065) wasselected for further directed evolution. The variant with SEQ ID NO:5076 was used for process development due to its high RebD to RebMactivity.

Example 64 Beta-1,3-ADP-Glycosyltransferase Variants of SEQ ID NO: 5066

In this Example, experiments for evolution and screening ofβ1,3-glycosyltransferase (β1,3GT) polypeptides derived from SEQ ID NO:5066 for improved glucosylation of steviol glycosides using in situsynthesized ADP-glucose are described. Directed evolution of the GTencoded by SEQ ID NO: 5065 (i.e., SEQ ID NO: 5066) was carried out byconstructing libraries of variant genes in which mutations associatedwith improved production identified during the development of thepresent invention were recombined. These libraries were then plated,grown, and screened using the high-throughput (HTP) assay describedbelow to provide a twelfth round (“Round 12”) of 40 engineered GTvariant polypeptides with glucosyltransferase activity towardADP-glucose and steviol glycosides.

HTP Assay for Glucose Transfer from Sucrose to ADP and then fromADP-Glucose to Stevioside and Rebaudioside D

Assays were performed on 96-well plates of cleared E. coli culturelysates expressing SEQ ID NO: 5066 variants. Pellets were lysed, and thelysate was cleared as described in Example 60, and then diluted 10× intopotassium phosphate buffer, pH 6. Assays of combinatorial libraries wereconducted with 10 μL lysate in 100 μL reactions and with 4 mM stevioside(>95% purity) or rebaudioside D substrate, 0.2 mM ADP (Sigma, >93%purity) co-substrate, 0.15 g/L SUS SFP SEQ ID NO: 1582, and 24 mMsucrose (cane sugar). The following reaction conditions were used: 50 mMpotassium phosphate buffer, pH 6, 55° C. in a Thermotron® titre-plateshaker with 300 RPM shaking for 2 h. Reactions were solubilized,quenched, and diluted as described in Example 63. Samples were analyzedby RapidFire-MS/MS as described in Example 31, Table 31.1.Glycosyltransferase variant polypeptides that produced rebaudiosideA/rebaudioside M from stevioside/rebaudioside D with in situ synthesizedADP-glucose at greater quantities than SEQ ID NO: 5066 were identified.The engineered polypeptides are listed in Table 64.1. Shake-flask scalecultures were grown, lysed, cleared, and lyophilized to powder asdescribed in Example 31 for analysis of variants shown in Table 64.2relative to SEQ ID NO: 5066.

TABLE 64.1 β1,3GT Round 12 Variants and RebA and RebM Levels SEQ ID NO:Amino Acid Differences Increased Increased (nt/aa) (Relative to SEQ IDNO: 5066) RebA^(a) RebM^(a) 5261/5262 I198E/G259E − +++ 5263/5264I198D/R234Q + ++ 5265/5266 Q159N/G259E/R411T + + 5267/5268R881/Q159N/I198D − ++ 5269/5270 G259E + ++ 5271/5272 R88I/A110D/R234T −++ 5273/5274 I198D + +++ 5275/5276 I198E/K200S + ++ 5277/5278 R88I − ++5279/5280 R88I/I198D/G259E − +++ 5281/5282 R88I/I198D/K200S/G259E + ++5283/5284 R88I/A110D − ++ 5285/5286 R88I/G259N − ++ 5287/5288R88I/A110D/Q159N/I198D/R234T/G259N/Q265A − ++ 5289/5290R88I/A110D/Q159N/I198D/G259E + +++ 5291/5292 R88I/G259E/Q265A − ++5293/5294 Q159N/I198E/R411T − ++ 5295/5296 A110D/G259N/Q265A/R411T + +5297/5298 R88I/A110D/Q159N/I198E/K200S − +++ 5299/5300 R88I/A110D/I198S− +++ 5301/5302 R88I/A110D/I198D − +++ 5303/5304 R234E + − 5305/5306R88I/I198S − ++ 5307/5308 I198D/K200S/R234E + − 5309/5310 V3201/V428K +++ 5311/5312 K208E/V320I/V428K + ++ 5313/5314 K171E/K208E/V320I + +++5315/5316 K171P/Q177P + ++ 5317/5318 L174E/V320I + ++ 5319/5320K171P/L174E/E175N/Q177P/K208W/V320I/V428K/K439D + − 5321/5322 V428K + −5325/5326 L174E/E175N/Q177P/K208E/V320I/V428K − ++ 5327/5328K171E/K208E/V428K + ++ 5329/5330 K171E/Q177P/E226T/V428K/K439D + −5331/5332 K171P/E175N/Q177P/K208L/V320I/V428K + + 5333/5334 K208E/V428K− ++ 5335/5336 K171E/K208E/V320I/V428K + +++ 5337/5338L174E/E175N/V428K + − 5339/5340 K208E + + 5341/5342K208L/V320I/R331C/V428K + − ^(a)Levels of increased production weredetermined relative to the reference polypeptide of SEQ ID NO: 5066, anddefined as follows: “−“ = production less than that of the referencepolypeptide; “+” = production at least that of the referencepolypeptide, but less than 1.35-fold; “++” = at least 1.35-fold, butless than 1.8-fold; and “+++” = at least 1.8-fold increased production,as compared to the reference polypeptide.SFP Characterization Assay and Analysis for Glucosyl Transfer fromSucrose to ADP and then from ADP-Glucose to (Stevioside and RebaudiosideD

Shake flask powders (SFP) were reconstituted to a concentration of 20g/Land diluted to 0.006-0.2 g/L SFP in 100 μL total reaction volumes of50 mM potassium phosphate buffer, pH6, 4 mM stevioside (95% purity) orrebaudioside D, 0.4 mM ADP (Sigma, >93% purity) co-substrate, 0.15 g/LSUS SFP SEQ ID NO: 1582, and 24 mM sucrose (cane sugar). The reactionswere incubated at 55° C. in a Thermotron® titre-plate shaker with 300RPM shaking for 1 hour. The reactions were solubilized by 20× dilutionwith water, quenched by 5× dilution in acetonitrile with 0.2% formicacid, cleared by centrifugation, and diluted 5× with water for analysis.Glycosylated products were detected in by SPE-QQQ as described inExample 31, Table 31.1.

TABLE 64.2 β1,3GT Round 12 Shake Flask Powder Variants and RebA and RebMLevels SEQ ID NO: Amino Acid Differences Increased Increased (nt/aa)(Relative to SEQ ID NO: 5066) RebA^(a) RebM^(a) 5279/5280R88I/I198D/G259E − ++ 5289/5290 R88I/A110D/Q159N/I198D/G259E − +++5301/5302 R88I/A110D/I198D − ++ 5313/5314 K171E/K208E/V320I + +5323/5324 V320I/V428K + + ^(a)Levels of increased production weredetermined relative to the reference polypeptide of SEQ ID NO: 5066, anddefined as follows: “−” = production less than 0.95-fold; “+” =production at least 0.95-fold, but less than 1.2-fold; “++” = at least1.2-fold, but less than 1.9-fold; and “+++” = at least 1.9-foldincreased production, as compared to the reference polypeptide.

In these experiments, the six variants in Table 64.2 (i.e., SEQ ID NO:5280, 5290, 5302, 5314, 5324 produced rebaudioside A from steviosideand/or rebaudioside M from rebaudioside D with ADP-glucose at greaterquantities than SEQ ID NO: 5066. SEQ ID NO: 5290 performed best onrebaudioside D and had the most beneficial mutations. Thus, the encodingpolynucleotide (SEQ ID NO: 5289) was selected for further directedevolution.

Example 65 Beta-1,3-ADP-Glycosyltransferase Variants of SEQ ID NO: 5290

In this Example, experiments for evolution and screening ofβ1,3-glycosyltransferase (β1,3GT) polypeptides derived from SEQ ID NO:5290 for improved glucosylation of steviol glycosides using in situsynthesized ADP-glucose are described. Directed evolution of the GTencoded by SEQ ID NO: 5289 (i.e., SEQ ID NO: 5290) was carried out byconstructing libraries of variant genes in which mutations associatedwith improved production identified during the development of thepresent invention were recombined and in which certain structuralfeatures were subjected to saturation mutagenesis. These libraries werethen plated, grown, and screened using the high-throughput (HTP) assaydescribed below to provide a thirteenth round (“Round 13”) of 100engineered GT variant polypeptides with glucosyltransferase activitytoward ADP-glucose and steviol glycosides.

HTP Assay for Glucose Transfer from Sucrose to ADP and then fromADP-Glucose to Stevioside and Rebaudioside D

Assays were performed on 96-well plates of cleared E. coli culturelysates expressing SEQ ID NO: 5289 variants. Pellets were lysed, andlysate was cleared as described in Example 60, and then diluted 5× intopotassium phosphate buffer, pH 6. Assays were conducted with 10 μLlysate in 100 μL reactions and with 4 mM stevioside (>95% purity) orrebaudioside D substrate, 0.2 mM ADP (Sigma, >93% purity) co-substrate,0.15 g/L SUS SFP SEQ ID NO: 1764, and 24 mM sucrose (cane sugar). Thefollowing reaction conditions were used: 50 mM potassium phosphatebuffer, pH 6, 55° C. in a Thermotron® titre-plate shaker with 300 RPMshaking for 2 h. Reactions were solubilized, quenched, and diluted asdescribed in Example 33. Samples were analyzed by RapidFire-MS/MS asdescribed in Example 31, Table 31.1. Glycosyltransferase variantpolypeptides that produced rebaudioside A/rebaudioside M fromstevioside/rebaudioside D with in situ synthesized ADP-glucose atgreater quantities than SEQ ID NO: 5290 were identified. The engineeredpolypeptides are listed in Table 65.1. Shake-flask scale cultures weregrown, lysed, cleared, and lyophilized to powder as described in Example31 for analysis of variants shown in Table 65.2 relative to SEQ ID NO:5290.

TABLE 65.1 β1,3GT Round 13 Variants and RebA and RebM Levels SEQ ID NO:Amino Acid Differences Increased Increased (nt/aa) (Relative to SEQ IDNO: 5290) RebA^(a) RebM^(a) 5343/5344 R308L + + 5345/5346K53R/T54V/S55T/A153S/E201P + − 5349/5350 K53V/K171P/R308L/K392H + +5351/5352 I91T/E201P/K439D/Y444H + + 5353/5354 R308L/F327L/K439D ++ −5355/5356 E73S/W87M/E201P − ++ 5357/5358 K53V/T54V/R308L/K392H + −5359/5360 K247A/Y249I/S252E/S254M/V320I/V428K + − 5361/5362I25L/K247L/Y249I/S252E/G364S/V428K + − 5363/5364 V428K + + 5365/5366S253G/V320I + + 5367/5368 Y249I/S252E/S253G/S254M + + 5369/5370S252E/S253G/S254M + + 5371/5372 I25L/T61S/V428K + + 5373/5374 K190R + +5375/5376 T61S/K208W/S252E/S254M/V428K + − 5377/5378L107A/K247A/Y249I/S252E/S254M/G364S + + 5379/5380L107A/K208N/V320I/G364S/V428K ++ + 5381/5382 L107A ++ + 5383/5384K190R/K208N/K247L/S252E/V428K + + 5385/5386I25L/K208E/K247L/S252E/S253G/G364S/V428K + + 5387/5388 V320I + +5389/5390 V3201/V428K ++ + 5391/5392 I25L/T61S/K208N/S252E/S253G + −5393/5394 L107A/K247A/S252E + − 5395/5396T61S/L107T/K208N/S252E/S253G/S254M/G3645/V428K ++ + 5397/5398I25L/L107T/K208E/V320I/V428K + + 5399/5400 V320I/G3645/V428K + +5401/5402 L107A/K247A/V428K − + 5403/5404 Y249I/S252E/S254M/V428K + −5405/5406 V435Q ++ + 5407/5408 T172H ++ + 5409/5410 V435R ++ ++5411/5412 A427L + + 5413/5414 R296H ND + 5415/5416 K289S ND + 5417/5418Q177P ++ + 5419/5420 G404T ND + 5421/5422 A438Q ND + 5423/5424 D431END + 5425/5426 V428E/D434N ND + 5427/5428 T54A ND + 5429/5430 K171T ++ +5431/5432 W233Q/Q269R ND + 5433/5434 D431R ND + 5435/5436 E2L + +5437/5438 D347K ++ + 5439/5440 Q424W ND + 5441/5442 S106T ND + 5443/5444G169L + + 5445/5446 W233Q ND + 5447/5448 R417P ++ + 5449/5450 G169C ND +5451/5452 V179A + + 5453/5454 S317R ND + 5455/5456 V428I + + 5457/5458W233M + + 5459/5460 A251L ND + 5461/5462 V435K ND + 5463/5464 N408R ND +5465/5466 W233C + + 5467/5468 D347H + + 5469/5470 K171S + + 5471/5472R296Q + + 5473/5474 D300G ND + 5475/5476 G169V ND + 5477/5478 E2N + +5479/5480 V435M ND + 5481/5482 G169Q ND + 5483/5484 W233K ++ + 5485/5486E99P ND + 5487/5488 V428S ND + 5489/5490 V179S ND + 5491/5492 D347P ++ +5493/5494 R296A ND + 5495/5496 Q424A + + 5497/5498 W233V + + 5499/5500G169E + + 5501/5502 V435T ND + 5503/5504 K222A ND + 5505/5506 V428R + +5507/5508 V428Q ND + 5509/5510 W233L + + 5511/5512 N159L ND + 5513/5514V428F ND + 5515/5516 R38Q − + 5517/5518 F264A ++ + 5519/5520 D347R ++ +5521/5522 R109S ND + 5523/5524 T172N + + 5525/5526 V435C ND + 5527/5528K171V + + 5529/5530 A427R ++ + 5531/5532 F64S + ++ 5533/5534 E318T − +5535/5536 W233G + + 5537/5538 E259T ND + 5539/5540 T7P + + 5541/5542N68K ND + ^(a)Levels of increased production were determined relative tothe reference polypeptide of SEQ ID NO: 5290, and defined as follows:“−” = production less than that of the reference polypeptide; “+” =production at least that of the reference polypeptide, but less than1.3-fold; and “++” = at least 1.3-fold increased production, as comparedto the reference polypeptide.SFP Characterization Assay and Analysis for Glucosyl Transfer fromSucrose to ADP and then from ADP-Glucose to (Stevioside and RebaudiosideD

Shake flask powders (SFP) were reconstituted to a concentration of 20g/Land diluted to 0.006-0.2 g/L SFP in 100 μL total reaction volumes of50 mM potassium phosphate buffer, pH6, 4 mM stevioside (95% purity),rebaudioside D, or rebaudioside A (97% purity), 0.2 mM ADP (Sigma, >93%purity) co-substrate, 0.15 g/L SUS SFP SEQ ID NO: 1764, and 24 mMsucrose (cane sugar). The reactions were incubated at 50 and 55° C. in aThermotron® titre-plate shaker with 300 RPM shaking for 1 hour. Thereactions were solubilized by 20× dilution with water, quenched by 5×dilution in acetonitrile with 0.2% formic acid, cleared bycentrifugation, and diluted 5× with water for analysis. Glycosylatedproducts were detected in by SPE-QQQ as described in Example 31, Table31.1.

TABLE 65.2 β1,3GT Round 13 Shake Flask Powder Variants and RebA and RebMLevels SEQ ID NO: Amino Acid Differences Increased Increased (nt/aa)(Relative to SEQ ID NO: 5290) RebA^(a) RebM^(a) 5347/5348 W87M + +5355/5356 E73S/W87M/E201P + + 5363/5364 V428K + + 5371/5372I25L/T61S/V428K + + 5385/5386 I25L/K208E/K247L/S252E/S253G/ + +G364S/V428K ^(a)Levels of increased production were determined relativeto the reference polypeptide of SEQ ID NO: 5290, and defined as follows:“−” = production less than that of the reference polypeptide; and “+” =production at least that of the reference polypepide, but less than1.45-fold increased production, as compared to the referencepolypeptide.

In these experiments, the five variants in Table 65.2 (i.e., SEQ ID NO:5348, 5356, 5364, 5372, and 5386) produced rebaudioside A fromstevioside and/or rebaudioside M from rebaudioside D with ADP-glucose atgreater quantities than SEQ ID NO: 5290. SEQ ID NO: 5372 was selectedfor further directed evolution.

Example 66 Beta-1,3-ADP-Glycosyltransferase Variants of SEQ ID NO: 5372

In this Example, experiments for evolution and screening ofβ1,3-glycosyltransferase (β1,3GT) polypeptides derived from SEQ ID NO:5372 for improved glucosylation of steviol glycosides using in situsynthesized ADP-glucose are described. Directed evolution of the GTencoded by SEQ ID NO: 5371 (i.e., SEQ ID NO: 5372) was carried out byconstructing libraries of variant genes in which mutations associatedwith improved production identified during the development of thepresent invention were recombined and in which certain structuralfeatures were subjected to saturation mutagenesis. These libraries werethen plated, grown, and screened using the high-throughput (HTP) assaydescribed below to provide a fourteenth round (“Round 14”) of 74engineered GT variant polypeptides with glucosyltransferase activitytoward ADP-glucose and steviol glycosides.

HTP Assay for Glucose Transfer from Sucrose to ADP and then fromADP-Glucose to Stevioside and Rebaudioside D

Assays were performed on 96-well plates of cleared E. coli culturelysates expressing SEQ ID NO: 5371 variants. Pellets were lysed, and thelysate was cleared as described in Example 60, and then diluted 10× intopotassium phosphate buffer, pH 6. Assays were conducted with 10 μLlysate in 100 μL reactions and with 10 mM stevioside (>95% purity) orrebaudioside D substrate, 0.2 mM ADP (Sigma, >93% purity) co-substrate,0.15 g/L SUS SFP SEQ ID NO: 1764, and 40 mM sucrose (cane sugar). Thefollowing reaction conditions were used: 50 mM potassium phosphatebuffer, pH 6, 60° C. in a Thermotron® titre-plate shaker with 300 RPMshaking for 2 h. Reactions were solubilized, quenched, and diluted to˜10 μM steviol glycosides as described in Example 60. Samples wereanalyzed by RapidFire-MS/MS as described in Example 31, Table 31.1.Glycosyltransferase variant polypeptides that produced rebaudiosideA/rebaudioside M from stevioside/rebaudioside D with in situ synthesizedADP-glucose at greater quantities than SEQ ID NO: 5372 were identified.The engineered polypeptides are listed in Table 66.1. Shake-flask scalecultures were grown, lysed, cleared, and lyophilized to powder asdescribed in Example 1 for analysis of variants shown in Table 66.2relative to SEQ ID NO: 5372.

TABLE 66.1 β1,3GT Round 14 Variants and RebA and RebM Levels SEQ ID NO:Amino Acid Differences Increased Increased (nt/aa) (Relative to SEQ IDNO: 5372) RebM^(a) RebA^(a) 5543/5544 W87M/Q177P/Y249I +++ ++ 5545/5546I88R/K208E/Y249I/V320I − ++ 5547/5548 W87M/I88R/Q177P/D198E/Y249I +++ +5549/5550 W87M/I88R +++ + 5551/5552 N159Q/D198I/K208E/V320I +++ +++5553/5554 W87M/D198E/A199K ++ ++ 5555/5556 W87M/D198E +++ ++ 5557/5558I88R/V320I + +++ 5559/5560 W87M +++ ++ 5561/5562W87M/I88R/E94K/N159Q/Q177P/ +++ ++ K208E 5563/5564 W87M/K208E +++ ++5565/5566 I88R/Q177P/K208E + ++ 5567/5568 I88R/D198I − ++ 5569/5570W87M/I88R/K208E ++ + 5571/5572 I88R +++ ++ 5573/5574W87M/I88R/N159Q/D198E +++ + 5575/5576 W87M/K208E/V320I ++ + 5577/5578Q177P/D198E +++ ++ 5579/5580 Q177P/K208E ++ ++ 5581/5582N159Q/Q177P/K208E +++ ++ 5583/5584 W87M/N159Q/Q177P +++ ++ 5585/5586I88R/Q177P/V320I ++ ++ 5587/5588 W87M/N159Q/V320I ++ + 5589/5590N159Q/Q177P/D198E +++ ++ 5591/5592 W87M/V320I +++ + 5593/5594 I88R/A199K− ++ 5595/5596 I88R/A199K/K208E − ++ 5597/5598 W87M/D198E/K208E/V320I+++ ++ 5599/5600 I88R/K208E − ++ 5601/5602 I88R/Q177P ++ +++ 5603/5604P272K ND + 5605/5606 D268G ND + 5607/5608 P158D ND + 5609/5610 E2H ND +5611/5612 E2G ND + 5613/5614 E2P ND + 5615/5616 S374R ND + 5617/5618S135A ND + 5619/5620 K171T ND + 5621/5622 S214L ND + 5623/5624 R173NND + 5625/5626 L256P ND + 5627/5628 L437T ND + 5629/5630 L257Q ND +5631/5632 L437I ND ++ 5633/5634 K222R ND + 5635/5636 D134S ND +5637/5638 S374K ND ++ 5639/5640 N408D ND + 5641/5642 P158E ND +5643/5644 E113S ND + 5645/5646 K392D ND + 5647/5648 Y453R ND + 5649/5650K439A ND + 5651/5652 N195H ND + 5653/5654 K302R ND + 5655/5656 L257AND + 5657/5658 R412H ND + 5659/5660 E330D ND + 5661/5662 R173P ND +5663/5664 N399D ND + 5665/5666 A348C ND + 5667/5668 D134G ND + 5669/5670K289T ND + 5671/5672 S253G ND + 5673/5674 D300G ND + 5675/5676 E73P ND +5677/5678 F49A ND + 5679/5680 D268H ND ++ 5681/5682 D268A ND + 5683/5684E445T ND + 5685/5686 E73T ND + 5687/5688 R411Q ND + 5689/5690 L437V ND++ ^(a)Levels of increased production were determined relative to thereference polypeptide of SEQ ID NO: 5372, and defined as follows: “−” =production less than 0.99-fold; “+” = production at least 0.99-fold, butless than 1.25-fold; “++” = at least 1.25-fold, but less than 1.6-fold;and “+++” = at least 1.6-fold increased production, as compared to thereference polypeptide. ND = not determined.SFP Characterization Assay and Analysis for Glucosyl Transfer fromSucrose to ADP and then from ADP-Glucose to Stevioside and RebaudiosideD

Shake flask powders (SFP) were reconstituted to a concentration of 20g/L and diluted to 0.006-0.2 g/L SFP in 100 μL total reaction volumes of50 mM potassium phosphate buffer, pH6, 4 mM stevioside (95% purity),rebaudioside D, rebaudioside E, or rebaudioside A (97% purity), 0.2 mMADP (Sigma, >93% purity) co-substrate, 0.15 g/L SUS SFP SEQ ID NO: 1764,and 24 mM sucrose (cane sugar). The reactions were incubated at 55 and60° C. in a Thermotron® titre-plate shaker with 300 RPM shaking for 2hours. The reactions were solubilized by 20× dilution with water,quenched by 5× dilution in acetonitrile with 0.2% formic acid, clearedby centrifugation, and diluted 5× with water for analysis. Glycosylatedproducts were detected in by SPE-QQQ as described in Example 31, Table31.1.

TABLE 66.2 β1,3GT Round 14 Shake Flask Powder Variants and RebA, RebI,and RebM Levels Amino Acid Differences Increased Increased SEQ ID NO:(Relative to Increased Increased RebE to RebD to (nt/aa) SEQ ID NO:5372) RebA^(a) RebI^(a) rebM^(a) rebM^(a) 5551/5552N159Q/D198I/K208E/V320I + + ++ + 5561/5562W87M/I88R/E94K/N159Q/Q177P/ + + +++ ++ K208E 5573/5574W87M/I88R/N159Q/D198E + + +++ + 5577/5578 Q177P/D198E + + ++ + 5581/5582N159Q/Q177P/K208E + + +++ + 5589/5590 N159Q/Q177P/D198E + + +++ +^(a)Levels of increased production were determined relative to thereference polypeptide of SEQ ID NO: 5372, and defined as follows: “−” =production less than that of the reference polypeptide; “+” = productionat least that of the reference polypeptide, but less than 2-fold; “++” =at least 2-fold, but less than 4-fold; and “+++” = at least 4-foldincreased production, as compared to the reference polypeptide.

In these experiments, the six variants in Table 66.2 (i.e., SEQ ID NO:5552, 5562, 6675, 5578, 5582, and 5590) produced more rebaudioside Afrom stevioside, less rebaudioside I from rebaudioside A, morerebaudioside M from rebaudioside E, and/or rebaudioside M fromrebaudioside D with ADP-glucose at greater quantities than SEQ ID NO:5372. SEQ ID NO: 5562 was the most improved for all four reactions, soit was selected for further directed evolution.

Example 67 Beta-1,3-ADP-Glycosyltransferase Variants of SEQ ID NO: 5562

In this Example, experiments for evolution and screening ofβ1,3-glycosyltransferase (β1,3GT) polypeptides derived from SEQ ID NO:5562 for improved glucosylation of steviol glycosides using in situsynthesized ADP-glucose are described. Directed evolution of the GTencoded by SEQ ID NO: 5561 (i.e., SEQ ID NO: 5562) was carried out byconstructing libraries of variant genes in which mutations associatedwith improved activity identified during the development of the presentinvention were recombined and in which certain structural features weresubjected to saturation mutagenesis. These libraries were then plated,grown, and screened using the high-throughput (HTP) assay describedbelow to provide a fifteenth round (“Round 15”) of 62 engineered GTvariant polypeptides with glucosyltransferase activity towardADP-glucose and steviol glycosides.

HTP Assay for Glucose Transfer from Sucrose to ADP and then fromADP-Glucose to Stevioside and Rebaudioside D

Assays were performed on 96-well plates of cleared E. coli culturelysates expressing SEQ ID NO: 5562 variants. Pellets were lysed, andlysate was cleared as described in Example 60 and then diluted 5× into50 mM potassium phosphate buffer, pH 6. Assays were conducted with 10 μLlysate in 100 μL reactions and with 10 mM stevioside (>95% purity) orrebaudioside D substrate, 0.1-0.2 mM ADP (Sigma, >93% purity)co-substrate, 0.15 g/L SUS SFP SEQ ID NO: 1764, and 24-40 mM sucrose(cane sugar). The following reaction conditions were used: 50 mMpotassium phosphate buffer, pH 6, 50 or 60° C. in a Thermotron®titre-plate shaker with 300 RPM shaking for 2 h. Reactions weresolubilized, quenched, and diluted to ˜10 μM steviol glycosides asdescribed in Example 60. Samples were analyzed by RapidFire-MS/MS asdescribed in Example 31, Table 31.1. Glycosyltransferase variantpolypeptides that produced rebaudioside A/rebaudioside M fromstevioside/rebaudioside D with in situ synthesized ADP-glucose atgreater quantities than SEQ ID NO: 5562 were identified. The engineeredpolypeptides from the combinatorial libraries are listed in Table 67.1,and those from the saturation mutagenesis libraries are listed in Table67.2. Shake-flask scale cultures were grown, lysed, cleared, andlyophilized to powder as described in Example 31 for analysis ofvariants shown in Table 67.3 relative to SEQ ID NO: 5562.

TABLE 67.1 β1,3GT Round 15 Combinatorial Variants and RebA Levels SEQ IDNO: Amino Acid Differences Increased (nt/aa) (Relative to SEQ ID NO:5562) RebA^(a) 5691/5692 F64S/G169E/E201P/D347P/K392H + 5693/5694K171T/E201P/K392H/R417P +++ 5695/5696G169E/K171T/T172H/F264A/K392H/V435Q + 5697/5698G169E/T172H/E201P/F264A/D347G ++ 5699/5700 I91T/E201P/F264A/D347G/K392H++ 5701/5702 G169E/T172H/E201P/D347G/K392H ++ 5703/5704G169E/K171T/E201P/F264A/K392H/V435Q + 5705/5706E201P/F264A/D347P/K392H/V435Q ++ 5707/5708G169E/T172H/E201P/F264A/D347K/K392H/V435R ++ 5709/5710E201P/F264A/D347K/K392H/R417P/V435R + 5711/5712G169E/T172H/E201P/R417P/V435R + 5713/5714F64S/T172H/F264A/D268V/D347P/K392H/R417P + 5715/5716E201P/F264A/D347K/R417P/V435R ++ 5717/5718I91T/K94E/K171T/T172H/E201P/F264A/D347G + 5719/5720K171T/T172H/E201P/F264A/K392H/R417P/V435R +++ 5721/5722K94E/E201P/F264A/D347G/V435Q + 5723/5724 L107A/S252E/S317R + 5725/5726W233K/S252E/V320I +++ 5727/5728 W233K/S252E/S317R/N408R ++ 5729/5730K190R/W233K/S252E/V3201/N408R ++ 5731/5732L107A/K190R/W233K/S252E/S317R/V320I +++ 5733/5734W233K/S252E/S317R/A427R ++ 5735/5736T7P/K190R/W233K/S252E/S317R/V320I/A427R/K428R +++ 5737/5738T7P/L107A/W233K/S252E/S253G + 5739/5740 W233K/S252E/V320I/N408R/K428R ++5741/5742 T7P/W233K/S252E/S253G/N408R +++ 5743/5744W233K/S252E/S253G/N408R/A427L ++ 5745/5746T7P/K190R/W233K/S252E/S253G/V320I/A427L + 5747/5748T7P/W233K/S252E/S253G/S317R/N408R/A427L +++ 5749/5750T7P/L107A/W233K/S252E/S317R/N408R + 5751/5752T7P/L107A/K190R/W233K/S252E/S253G/S317R/N408R/A427R ++ 5753/5754T7P/W233K/S252E/S317R/V320I/A427L + 5755/5756E2N/T7P/L107A/W233K/S252E/S253G/V320I + 5757/5758K190R/W233K/S252E/S253G/S317R/V320I/N408R/K428R + 5759/5760E2L/T7P/W233K/S252E/S253G/V320I/A427R/K428R + 5761/5762W233K/S252E/S253G/S317R/V320I/A427R/K428R +++ 5763/5764T7P/W233K/S252E/S317R/K428R + 5765/5766 E2N/W233K/S252E/S317R/V320I +5767/5768 L107A/W233K/S252E/S253G ++ 5769/5770T7P/L107A/W233K/S252E/S317R/V320I + 5771/5772T7P/K190R/W233K/S252E/N408R/A427R + 5773/5774 K190R/W233K/S252E ++5775/5776 L107A/K190R/W233K/S252E + 5777/5778K190R/W233K/S252E/S317R/N408R +++ 5779/5780E2N/T7P/L107A/VV233K/S252E/S253G/V320I/N408R/K428R + 5781/5782A348S/S374R/V435R + 5783/5784 S374R + ^(a)Levels of increased productionwere determined relative to the reference polypeptide of SEQ ID NO:5562, and defined as follows: “+” = production at least 1.59-fold, butless than 2.8-fold; “++” = at least 2.8-fold, but less than 3.1-fold;and “+++” = at least 3.1-fold increased production, as compared to thereference polypeptide.

TABLE 67.2 β1,3GT Round 15 Saturation Mutagenesis Variants and RebA andRebM Levels Amino Acid Differences Increased Increased Increased SEQ IDNO: (Relative to RebA, RebA, RebM, (nt/aa) SEQ ID NO: 5562) 50° C.^(a)60° C.^(a) 60° C.^(a) 5785/5786 L56A − + ++ 5787/5788 S55V − − +5789/5790 R308Q − ++ +++ 5791/5792 K336Q + − − 5793/5794 G364A + +++ +++5795/5796 E342W + + + 5797/5798 T282S ++ +++ +++ 5799/5800 R308L + +++++ 5801/5802 G364S +++ +++ +++ 5803/5804 E407C + + +++ 5805/5806 L391C +++ +++ 5807/5808 E407V ++ ++ +++ 5809/5810 V14I ++ ++ ++ 5811/5812S255L + + + 5813/5814 M422Q − + + ^(a)Levels of increased productionwere determined relative to the reference polypeptide of SEQ ID NO:5562, and defined as follows: “−” = production less than that of thereference polypeptide; “+” = production at least that of the referencepolypeptide, but less than 1.25-fold; “++” = at least 1.25-fold, butless than 1.4-fold; and “+++” = at least 1.4-fold increased production,as compared to the reference polypeptide.SFP Characterization Assay and Analysis for Glucosyl Transfer fromSucrose to ADP and then from ADP-Glucose to Stevioside and RebaudiosideD

Shake flask powders (SFP) were reconstituted to a concentration of 20g/Land diluted to 0.006-0.2 g/L SFP in 100 μL total reaction volumes of50 mM potassium phosphate buffer, pH6, 10 mM stevioside (95% purity) orrebaudioside D, 0.2 mM ADP (Sigma, >93% purity) co-substrate, 0.15 g/LSUS SFP SEQ ID NO: 1764, and 24 mM sucrose (cane sugar). The reactionswere incubated at 50 and 60° C. in a Thermotron® titre-plate shaker with300 RPM shaking for 2 hours. The reactions were solubilized by 20×dilution with water, quenched by 5× dilution in acetonitrile with 0.2%formic acid, cleared by centrifugation, and diluted 5× with water foranalysis. Glycosylated products were detected in by SPE-QQQ as describedin Example 31, Table 31.1.

TABLE 67.3 β1,3GT Round 15 Shake Flask Powder Variants and RebA and RebMLevels Increased Increased Increased SEQ ID NO: Amino Acid DifferencesRebA, RebA, RebM, (nt/aa) (Relative to SEQ ID NO: 5562) 50° C.^(a) 60°C.^(a) 60° C.^(a) 5693/5694 K171T/E201P/K392H/R417P + + ++ 5707/5708G169E/T172H/E201P/F264A/D347K/K392H/ + ++ +++ V435R 5713/5714F64S/T172H/F264A/D268V/D347P/K392H/ + + +++ R417P 5719/5720K171T/T172H/E201P/F264A/K392H/R417P/ + ++ ++ V435R 5731/5732L107A/K190R/W233K/S252E/S317R/V320I − + +++ 5735/5736T7P/K190R/W233K/S252E/S317R/V320I/ − ++ ++ A427R/K428R 5761/5762W233K/S252E/S253G/S317R/V320I/A427R/ − + ++ K428R 5777/5778K190R/W233K/S252E/S317R/N408R − + +++ ^(a)Levels of increased productionwere determined relative to the reference polypeptide of SEQ ID NO:5562, at 0.025 g/L shake flask powder and defined as follows: “−” =production less than that of the reference polypeptide; “+” = productionat least that of the reference polypeptide, but less than 2-fold; “++” =at least 2-fold, but less than 2.4-fold; and “+++” = at least 2.4-foldincreased production, as compared to the reference polypeptide.

In these experiments, the eight variants in Table 67.3 produced morerebaudioside A from stevioside and/or more rebaudioside M fromrebaudioside D at 50° C. and/or 60° C. than SEQ ID NO: 5562. SEQ ID NO:5708 was the most improved for converting stevioside to rebaudioside Afor both temperatures and also improved for converting rebaudioside D torebaudioside M at 60° C., so it was selected for further directedevolution.

Example 68 Beta-1,3-ADP-Glycosyltransferase Variants of SEQ ID NO: 5708

In this Example, experiments for evolution and screening ofβ1,3-glycosyltransferase (β1,3GT) polypeptides derived from SEQ ID NO:5708 for improved glucosylation of steviol glycosides using in situsynthesized ADP-glucose are described. Directed evolution of the GTencoded by SEQ ID NO: 5707 (i.e., SEQ ID NO: 5708) was carried out byconstructing libraries of variant genes in which mutations associatedwith improved production identified during the development of thepresent invention were recombined. These libraries were then plated,grown, and screened using the high-throughput (HTP) assay describedbelow to provide a sixteenth round (“Round 16”) of 100 engineered GTvariant polypeptides with glucosyltransferase activity towardADP-glucose and steviol glycosides.

HTP Assay for Glucose Transfer from Sucrose to ADP and then fromADP-Glucose to Stevioside and Rebaudioside D

Assays were performed on 96-well plates of cleared E. coli culturelysates expressing SEQ ID NO: 5708 variants. Pellets were lysed, andlysate was cleared as described in Example 60, and then diluted 20× into50 mM potassium phosphate buffer, pH6. Assays were conducted with 10 μLlysate in 100 μL reactions and with 10 mM stevioside (>95% purity),rebaudioside D, rebaudioside A (>97% purity), or rebaudioside Esubstrate, 0.2 mM ADP (Sigma, >93% purity) co-substrate, 0.15 g/L SUSSFP SEQ ID NO: 1764, and 24m sucrose (cane sugar). The followingreaction conditions were used: 50 mM potassium phosphate buffer, pH 6,60° C. in a Thermotron® titre-plate shaker with 300 RPM shaking for 3h.Reactions were solubilized, quenched, and diluted to ˜10 μM steviolglycosides as described in Example 60. Samples were analyzed byRapidFire-MS/MS as described in Example 31, Table 31.1.Glycosyltransferase variant polypeptides that produced rebaudioside Arebaudioside M from stevioside/rebaudioside D with in situ synthesizedADP-glucose at greater quantities than SEQ ID NO: 5708 were identified.The engineered polypeptides are listed in Table 68.1. Shake-flask scalecultures were grown, lysed, cleared, and lyophilized to powder asdescribed in Example 31 for analysis of variants shown in Table 68.2relative to SEQ ID NO: 5708.

TABLE 68.1 β1,3GT Round 16 Combinatorial Variants and RebA Levels SEQ IDNO: Amino Acid Differences Increased (nt/aa) (Relative to SEQ ID NO:5708) RebA^(a) 5815/5816 R173N/K190R/S252E/L257A/K347G/A427R/ +++ L437I5817/5818 R173N + 5819/5820 W233K/S252E/L437V +++ 5821/5822R173N/K190R/L257A/A427R/L437I ++ 5823/5824 K347G/R411Q/L437I ++5825/5826 W233K/S252E/E285Q/L437V ++ 5827/5828 R173N/S252E +++ 5829/5830E285Q/K347G/L437V + 5831/5832 R173N/S252E/D268H/L437V ++ 5833/5834L257A/K347G/R411Q/L437V + 5835/5836 S252E/L257A/K347G/L437V ++ 5837/5838R173N/S252E/K347G/R411Q ++ 5839/5840 W233K/S252E +++ 5841/5842R173N/W233K/S252E/L257A/A427R + 5843/5844W233K/S252E/L257A/A427R/L437V + 5845/5846 W233K/S252E/R411Q/L437V +++5847/5848 R173N/L257A/S374K/L437V + 5849/5850K190R/S252E/L257A/R411Q/L437I + 5851/5852R173N/K190R/L257A/S374K/L437V + 5853/5854 W233K/S374K/L437I + 5855/5856K347G/A427R/L437V ++ 5857/5858 K190R/W233K/S252E/L257A/K347G/R411Q/ ++L437I 5859/5860 W233K/S252E/L257A/K347G +++ 5861/5862W233K/S252E/L257A/K347G/R411Q/L437I +++ 5863/5864K190R/S252E/L257A/E285Q/A427R ++ 5865/5866R173N/K190R/W233K/S252E/A427R/L437V + 5867/5868 S374K/R411Q/L437V +5869/5870 R173N/W233K/L437I + 5871/5872 R173N/A427R/L437I + 5873/5874S374K/R411Q/L437I + 5875/5876 K190R/S252E ++ 5877/5878 S252E/L257A ++5879/5880 W233K/S374K + 5881/5882 R411Q/L437V + 5883/5884 S374K +5885/5886 K190R/S374K/A427R/L437V + 5887/5888 L437I ++ 5889/5890R173N/S374K/L437I ++ 5891/5892 K190R/S252E/L257A/A427R + 5893/5894W233K/S252E/L257A ++ 5895/5896 W233K/S252E/E285Q + 5897/5898L163A/L257Q/K302R/G364S ++ 5899/5900 K302R/G364S ++ 5901/5902E2P/K4T/P158D/L163A/G364S/S449R +++ 5903/5904 E2P/P158D/L163R +5905/5906 E2P/L163R/G364S/Y453R + 5907/5908 E2P/K4T/L163A/L257Q ++5909/5910 G364S/N399D ++ 5911/5912 E2P/K4T/L163A/G364S/Y453R ++5913/5914 E2P/K4T/L163A/E330D/S449R ++ 5915/5916K4T/E113S/P158D/K302R/E330D/G364S + 5917/5918 E2P/E113S/S449R +5919/5920 E2P/K4T/K302R + 5921/5922 L163R/K302R/E330D/G364S/S449R +5923/5924 E330D/G364S + 5925/5926 E2P/K4T/E113S/P158D/L163A/E330D/S449R+++ 5927/5928 E2P/P158D/L163A/G364S/N399D +++ 5929/5930E2P/K4T/L163R/L257Q/E330D/N399D/S449R ++ 5931/5932E2P/E113S/L163R/S449R + 5933/5934 L163A/G364S ++ 5935/5936P158D/L163A/G364S ++ 5937/5938 E2P/K4T/P158D/E330D +++ 5939/5940E2P/E113S + 5941/5942 E2P/K4T/L163R/K222R/K302R/E330D/G364S/ + S449R5943/5944 E113S/P158D/L163A/G3645/N399D + 5945/5946K4T/E113S/L163A/G364S/N399D ++ 5947/5948E2P/K4T/E113S/P158D/L163R/K302R/G364S/ + N399D/S449R 5949/5950E2P/K4T/L163A/G3645 +++ 5951/5952 K4T/P158D/L163A/G3645/N399D ++5953/5954 E2P/E113S/L163A + 5955/5956E2P/K4T/P158D/K222R/L257Q/K302R/E330D ++ 5957/5958K4T/E113S/P158D/L163A/E330D/G3645/ ++ N399D 5959/5960E2P/K4T/E113S/L163R/S449R + 5961/5962 E113S/P158D/L163R/G3645/N399D +5963/5964 P158D/L163R/K302R/E330D/G3645/N399D + 5965/5966K4T/G3645/5449R ++ 5967/5968 E2P/G3645/5449R +++ 5969/5970E2P/E113S/E330D/N399D + 5971/5972 P158D/L163A/L257Q/E330D/G364S/S449R +5973/5974 D134S + 5975/5976 L107A/N195H/R417P/K439P +++ 5977/5978N195H/P272K/V320I/K439P + 5979/5980 D134S/S135A/N195H/D268A/S317R +5981/5982 N195H/S317R/V320I + 5983/5984 L107A/N195H/P272K ++ 5985/5986L107A/S374T/R417P/K439P + 5987/5988L107A/D134S/S135A/N195H/R412H/R417P + 5989/5990L107A/N195H/D268A/P322L/K439P +++ 5991/5992 N408D/R417P + 5993/5994D268A/R417P + 5995/5996 M87W/W266L ++ 5997/5998 M87W/D198E + 5999/6000D198E/L292P +++ 6001/6002 M87W/H95L/D198E + 6003/6004 D198I +++6005/6006 M87W/P322S +++ 6007/6008 D198I/P244L ++ 6009/6010 M87W +6011/6012 D198E + 6013/6014 M87W/D198I +++ ^(a)Levels of increasedproduction were determined relative to the reference polypeptide of SEQID NO: 5708, and defined as follows: “+” = production at least1.05-fold, but less than 1.42-fold; “++” = at least 1.42-fold, but lessthan 1.6-fold; and “+++” = at least 1.6-fold increased production, ascompared to the reference polypeptide.SFP Characterization Assay and Analysis for Glucosyl Transfer fromSucrose to ADP and then from ADP-Glucose to Stevioside and RebaudiosideD

Shake flask powders (SFP) were reconstituted to a concentration of 20g/L and diluted to 0.006-0.2 g/L SFP in 100 μL total reaction volumes of50 mM potassium phosphate buffer, pH6, 10 mM stevioside (>95% purity),rebaudioside D, rebaudioside E, or rebaudioside A (>97% purity), 0.2 mMADP (Sigma, >93% purity) co-substrate, 0.15 g/L SUS SFP SEQ ID NO: 1764,and 24 mM sucrose (cane sugar). The reactions were incubated at 50° C.and 60° C. in a Thermotron® titre-plate shaker with 300 RPM shaking for3 hours. The reactions were solubilized by 20× dilution with water,quenched by 5× dilution in acetonitrile with 0.2% formic acid, clearedby centrifugation, and diluted 10× with water for analysis. Glycosylatedproducts were detected in by SPE-QQQ as described in Example 31, Table31.1.

TABLE 68.2 β1,3GT Round 16 Shake Flask Powder Variants and RebA and RebMLevels SEQ ID Increased Increased Increased Increased NO: Amino AcidDifferences RebA, RebA, RebM, RebM from E, (nt/aa) (Relative to SEQ IDNO: 5708) 50° C.^(a) 60° C.^(a) 60° C.^(a) 60° C.^(a) 5815/5816R173N/K190R/S252E/L257A/ − +++ − − K347G/A427R/L437I 5839/5840W233K/S252E − ++ − + 5845/5846 W233K/S252E/R411Q/L437V − + − − 5901/5902E2P/K4T/P158D/L163A/G364S/ ++ + + + S449R 5927/5928E2P/P158D/L163A/G364S/ ++ + + ++ N399D 5949/5950 E2P/K4T/L163A/G364S++ + + ND 5975/5976 L107A/N195H/R417P/K439P ++ ++ + ND 5983/5984L107A/N195H/P272K ++ + − ND 5989/5990 L107A/N195H/D268A/P322L/ +++ + −ND K439P 5997/5998 M87W/D198E +++ +++ − ND 6009/6010 M87W +++ +++ − ND6011/6012 D198E ++ + + ND ^(a)Levels of increased production weredetermined relative to the reference polypeptide of SEQ ID NO: 5708, at0.025 g/L shake flask powder and defined as follows: “−” = productionless than that of the reference polypeptide; “+” = production at leastthat of the reference polypeptide, but less than 1.26-fold; “++” = atleast 1.26-fold, but less than 1.5-fold; and “+++” = at least 1.5-foldincreased production, as compared to the reference polypeptide. ND = notdetermined.

In these experiments, the thirteen variants in Table 68.2 produced morerebaudioside A from stevioside and/or more rebaudioside M fromrebaudioside D at 50° C. and/or 60° C. than SEQ ID NO: 5708. SEQ ID NO:5976 had the greatest improvement for conversion of stevioside torebaudioside A without a loss in rebaudioside D to rebaudioside Mactivity for both temperatures, so this variant was selected for furtherdirected evolution.

Example 69 Beta-1,3-ADP-Glycosyltransferase Variants of SEQ ID NO: 5976

In this Example, experiments for evolution and screening ofβ1,3-glycosyltransferase (β1,3GT) polypeptides derived from SEQ ID NO:5976 for improved glucosylation of steviol glycosides using in situsynthesized ADP-glucose are described. Directed evolution of the GTencoded by SEQ ID NO: 5975 (i.e., SEQ ID NO: 5976) was carried out byconstructing libraries of variant genes in which mutations associatedwith improved production identified during the development of thepresent invention were recombined and in which certain structuralfeatures were subjected to saturation mutagenesis. These libraries werethen plated, grown, and screened using the high-throughput (HTP) assaydescribed below to provide a seventeenth round (“Round 17”) of 123engineered GT variant polypeptides with glucosyltransferase activitytoward ADP-glucose and steviol glycosides.

HTP Assay for Glucose Transfer from Sucrose to ADP and then fromADP-Glucose to Stevioside and Rebaudioside D

Assays were performed on 96-well plates of cleared E. coli culturelysates expressing SEQ ID NO: 5976 variants. Pellets were lysed, and thelysate was cleared as described in Example 60 and then diluted 20× intobuffer. In order to thermally challenge the lysates, they werepre-incubated in a thermocycler for 15 minutes at 65.5° C. Assays wereconducted with 10 μL pre-incubated lysate in 100 μL reactions and with10 mM stevioside (>95% purity) or rebaudioside D substrate, 0.2 mM ADP(Sigma, >93% purity) co-substrate, 0.1 g/L SUS SFP SEQ ID NO: 1804, and24 mM sucrose (cane sugar). The following reaction conditions were used:50 mM potassium phosphate buffer, pH 6, 60° C. in a Thermotron®titre-plate shaker with 300 RPM shaking for 4 h. Reactions weresolubilized by 20× dilution with water, quenched by 5× dilution inacetonitrile with 0.2% formic acid, and diluted 10× with water foranalysis. Samples were analyzed by RapidFire-MS/MS as described inExample 31, Table 31.1. Glycosyltransferase variant polypeptides thatproduced rebaudioside A/rebaudioside M from stevioside/rebaudioside Dwith in situ synthesized ADP-glucose at greater quantities than SEQ IDNO: 5976 were identified. The engineered polypeptides are listed inTable 69.1. Shake-flask scale cultures were grown, lysed, cleared, andlyophilized to powder as described in Example 31 for analysis ofvariants shown in Table 69.2 relative to SEQ ID NO: 5976.

TABLE 69.1 β1,3GT Round 17 Variants and RebA and RebM Levels SEQ ID NO:Amino Acid Differences Increased Increased (nt/aa) (Relative to SEQ IDNO: 5976) RebA^(a) RebM^(a) 6015/6016 K4T/D198E/N399D/E407V + +6017/6018 E2P/V14I/T282S/R308L ++ +++ 6019/6020T282S/R308L/E342W/G364A/S449R +++ +++ 6021/6022 E2P/V14I/R308L/G364A ++++++ 6023/6024 E2P/K4T/T282S/E342W/G364A/E407V +++ +++ 6025/6026K4T/L163A/T282S/R308L/E407V ++ ++ 6027/6028 E2P/T282S/N399D + ++6029/6030 P158D/T282S/E407V ++ + 6031/6032E2P/V14I/P158D/D198E/E407V/S449R +++ +++ 6033/6034 E2P/V14I/R308L ++ ++6035/6036 V14I/P158D/L163A/D198E/G364A ++ +++ 6037/6038E2P/K4T/V14I/T282S/R308L/G364A +++ +++ 6039/6040 D198E/T282S/E407V ++++++ 6041/6042 E2P/V14I/L163A/R308L/G364A +++ +++ 6043/6044E2P/R308L/G364A/N399D +++ ++ 6045/6046 E2P/V14I/T282S/N399D ++ ++6047/6048 T282S/R308L/S449R + + 6049/6050K4T/V14I/T282S/R308L/G364A/E407V/S449R +++ +++ 6051/6052E2P/K4T/V14I/D198E/R308L/E407V +++ ++ 6053/6054P158D/G364A/N399D/E407V/S449R ++ + 6055/6056V14I/T282S/E342W/G364A/L391C ++ ++ 6057/6058 E2P/T282S/R308L/E342W/E407V++ +++ 6059/6060 E2P/K4T/P158D/L163A/G364A/N399D/E407V ++ ++ 6061/6062L163A/T282S/N399D/E407V ++ + 6063/6064V14I/P158D/D198E/T282S/E342W/G364A/S449R +++ ++ 6065/6066P158D/T282S/E407V/S449R ++ ++ 6067/6068 E2P/K4T/V14I/P158D/T282S/E407V+++ +++ 6069/6070 V14I/L163A/D198E/T282S/E342W/G364A/S449R +++ ++6071/6072 E2P/L163A/T282S ++ + 6073/6074 E2P/T282S ++ + 6075/6076V14I/P158D/L163A/T282S/G364A +++ + 6077/6078E2P/K4T/V14I/T282S/R308L/E407V ++ + 6079/6080K4T/V14I/L163A/T282S/R308L/E342W/E407V/S449R ++ + 6081/6082E2P/L163A/T282S/R308L/G364A +++ ++ 6083/6084E2P/K4T/L163A/G364A/N399D/S449R ++ +++ 6085/6086 L163A/D198E/T282S/E342W++ ++ 6087/6088 V14I/P158D/G364A ++ + 6089/6090K4T/V14I/D198E/R308L/G364A +++ + 6091/6092 E2P/D198E/T282S/R308L/E342W+++ +++ 6093/6094 K4T/V14I/P158D/T282S/G364A/L391C/E407V +++ ++6095/6096 K4T/V14I/G364A/L391C + ++ 6097/6098 K4T/V14I/L163A/T282S + +6099/6100 E2P/K4T/V14I/L163A/T282S/N399D ++ ++ 6101/6102 V14I/E407V ++++ 6103/6104 E2P/K4T/L163A/D198E/T282S/R308L/E342W ++ ++ 6105/6106L163A/T282S/E407V/S449R +++ +++ 6107/6108 E2P/P158D/L163A/E407V/S449R +++ 6109/6110 E2P/K4T/D198E/G364A/L391C/S449R ++ +++ 6111/6112 V14I/T282S++ ++ 6113/6114 V14I/L163A/T282S/R308L + ++ 6115/6116 E2P/R308L ++ +6117/6118 E2P/K4T/L163A/T282S/E342W/G364A +++ +++ 6119/6120 D198E/R308L++ + 6121/6122 E2P/T282S/R308L/L391C/E407V + + 6123/6124K4T/V14I/T282S + + 6125/6126 E2P/K4T/D198E/T282S/S449R +++ +++ 6127/6128E2P/K4T/V14I/T282S/G364A +++ +++ 6129/6130 E2P/V14I/T282S ++ + 6131/6132E2P/T282S/N399D/E407V +++ ++ 6133/6134 K4T/T282S/G364A/E407V +++ ++6135/6136 K4T/V14I/P158D/L163A/D198E/T282S/E407V +++ ++ 6137/6138E2P/K4T/P158D/D198E/T282S/G364A/E407V +++ +++ 6139/6140E2P/V14I/L163A/G364A/E407V +++ +++ 6141/6142V14I/T282S/N399D/E407V/S449R ++ ++ 6143/6144K4T/V14I/T282S/E342W/N399D/E407V + + 6145/6146E2P/V14I/T282S/R308L/G364A +++ ++ 6147/6148E2P/K4T/V14I/P158D/T282S/G364A/S449R +++ ++ 6149/6150E2P/D198E/T282S/N399D +++ ++ 6151/6152 P158D/G364A/S449R ++ + 6153/6154E2P/K4T/V14I/L163A/D198E ++ + 6155/6156 K4T/T282S/E342W/G364A/E407V+++ + 6157/6158 L163A/T282S/R308L/G364A/N399D +++ ++ 6159/6160D198E/T282S/R308L/E342W ++ + 6161/6162 V14I/T282S/G364A +++ ++ 6163/6164K4T/G364A ++ + 6165/6166 V14I/T282S/R308L + + 6167/6168V14I/T282S/G364A/L391C/E407V/S449R +++ ++ 6169/6170K4T/P158D/T282S/G364A/N399D ++ + 6171/6172 L75G + + 6173/6174 P139S + +6175/6176 L401F/E402L + ++ 6177/6178 P131V ND + 6179/6180 L138V ND +6181/6182 P90S ND + 6183/6184 S254Q + + 6185/6186 P139R + + 6187/6188M108H ND + 6189/6190 S106Y + + 6191/6192 R12S ND + 6193/6194 P90T ND +6195/6196 D416S + + 6197/6198 P139A + + 6199/6200 A427K ND + 6201/6202I44V ND + 6203/6204 V365I + + 6205/6206 S37R + + 6207/6208 K247C + ++6209/6210 D258N ND + 6211/6212 E115R + + 6213/6214 Q71L/R331K + +6215/6216 L75M + + 6217/6218 D416L + + 6219/6220 D114P + + 6221/6222D389S ND + 6223/6224 S456R ND + 6225/6226 S456* − + 6227/6228 S156C + +6229/6230 P70K + − 6231/6232 D389A + ++ 6233/6234 H248W + +++ 6235/6236A427R + ++ 6237/6238 L429W + + 6239/6240 K247L + + 6241/6242 A433L + +++6243/6244 E162A + + 6245/6246 H248L + +++ 6247/6248 K432L + ++ 6249/6250H248C + ++ 6251/6252 L174P + + 6253/6254 P112N ND + 6255/6256 D389E + ++6257/6258 P90Q − + 6259/6260 W74H + + ^(a)Levels of increased productionwere determined relative to the reference polypeptide of SEQ ID NO:5976, and defined as follows: “+” = production at least that of thereference polypeptide, but less than 2-fold; “++” = at least 2-fold, butless than 2.75-fold; and “+++” = at least 2.75-fold increasedproduction, as compared to the reference polypeptide.SFP Characterization Assay and Analysis for Glucosyl Transfer fromSucrose to ADP and then from ADP-Glucose to Stevioside and RebaudiosideD

Shake flask powders (SFP) were reconstituted to a concentration of 20g/L and diluted to 0.006-0.2 g/L SFP in 100 μL total reaction volumes of50 mM potassium phosphate buffer, pH6, 10 mM stevioside (>95% purity) orrebaudioside D, 0.2 mM ADP (Sigma, >93% purity) co-substrate, 0.1 g/LSUS SFP SEQ ID NO: 1804, and 24 mM sucrose (cane sugar). The reactionswere incubated at 55° C. with no pre-incubation or at 60° C. following15 minute pre-incubation at 65.5° C. in a Thermotron® titre-plate shakerwith 300 RPM shaking for 4 hours. The reactions were solubilized by 20×dilution with water, quenched by 5× dilution in acetonitrile with 0.2%formic acid, cleared by centrifugation, and diluted 10× with water foranalysis. Glycosylated products were detected in by SPE-QQQ as describedin Example 31, Table 31.1.

TABLE 69.2 β1, 3GT Round 17 Shake Flask Powder Variants and RebA andRebM Levels Increased Increased Increased SEQ ID NO: Amino AcidDifferences RebA, RebA, RebM, (nt/aa) (Relative to SEQ ID NO: 5976) 55°C^(a) 60° C^(a) 60° C^(a) 6147/6148 E2P/K4T/V14I/P158D/T282S/G364A/ + ++++ S449R 6037/6038 E2P/K4T/V14I/T282S/R308L/G364A + +++ ++ 6137/6138E2P/K4T/P158D/D198E/T282S/G364A/ + +++ ++ E407V 6091/6092E2P/D198E/T282S/R308L/E342W + + + 6067/6068E2P/K4T/V14I/P158D/T282S/E407V + ++ ++ 6049/6050K4T/V14I/T282S/R308L/G364A/E407V/ + +++ ++ S449R 6023/6024E2P/K4T/T282S/E342W/G364A/E407V + +++ ++ 6105/6106L163A/T282S/E407V/S449R + ++ ++ ^(a)Levels of increased production weredetermined relative to the reference polypeptide of SEQ ID NO: 5976, at0.025 g/L shake flask powder and defined as follows: “+” = production atleast 1.3-fold, but less than 2.4-fold; “++” = at least 2.4-fold, butless than 4-fold; and “+++” = at least 4-fold increased production, ascompared to the reference polypeptide.

In these experiments, the eight variants in Table 69.2 produced morerebaudioside A from stevioside and/or more rebaudioside M fromrebaudioside D at 55° C. and/or 60° C. than SEQ ID NO: 5976. SEQ ID NO:6138 had the greatest improvement for conversion of stevioside torebaudioside A under the conditions with pre-incubation followed by the60° C. assay, and was improved under the other conditions as well, sothis variant was selected for further directed evolution.

Example 70 Beta-1,3-ADP-Glycosyltransferase Variants of SEQ ID NO: 6138

In this Example, experiments for evolution and screening ofβ1,3-glycosyltransferase (β1,3GT) polypeptides derived from SEQ ID NO:6138 for improved glucosylation of steviol glycosides using in situsynthesized ADP-glucose are described. Directed evolution of the GTencoded by SEQ ID NO: 6137 (i.e., SEQ ID NO: 6138) was carried out byconstructing libraries of variant genes in which mutations associatedwith improved production identified during the development of thepresent invention were recombined and in which certain structuralfeatures were subjected to saturation mutagenesis. These libraries werethen plated, grown, and screened using the high-throughput (HTP) assaydescribed below to provide an eighteenth round (“Round 18”) of 100engineered GT variant polypeptides with glucosyltransferase activitytoward ADP-glucose and steviol glycosides.

HTP Assay for Glucose Transfer from Sucrose to ADP and then fromADP-Glucose to Stevioside and Rebaudioside D

Assays were performed on 96-well plates of cleared E. coli culturelysates expressing SEQ ID NO: 6137 variants. Pellets were lysed, andlysate was cleared as described in Example 60, and then diluted 20-40×into 50 mM potassium phosphate buffer, pH 6. In order to thermallychallenge the lysates, they were pre-incubated in a thermocycler for 15minutes at 68.6° C. Assays were conducted with 10 μL pre-incubatedlysate in 100 μL reactions and with 10 mM stevioside (>95% purity) orrebaudioside D substrate, 0.2 mM ADP (Sigma, >93% purity) co-substrate,0.1 g/L SUS SFP SEQ ID NO:1840, and 24 mM sucrose (cane sugar). Thefollowing reaction conditions were used: 50 mM potassium phosphatebuffer, pH6, 60° C. in a Thermotron® titre-plate shaker with 300 RPMshaking for 4h. Reactions were solubilized by 20× dilution with water,quenched by 5× dilution into acetonitrile with 0.2% formic acid, anddiluted 10× with water for analysis.

Samples were analyzed by RapidFire-MS/MS as described in Example 31,Table 31.1. Glycosyltransferase variant polypeptides that producedrebaudioside A rebaudioside M from stevioside/rebaudioside D with insitu synthesized ADP-glucose at greater quantities than SEQ ID NO: 6138were identified. The engineered polypeptides are listed in Table 70.1.Shake-flask scale cultures were grown, lysed, cleared, and lyophilizedto powder as described in Example 31 for analysis of variants shown inTable 70.2 relative to SEQ ID NO: 6138.

TABLE 70.1 β1, 3GT Round 18 Variants and RebA and RebM Levels SEQ ID NO:Amino Acid Differences Increased Increased (nt/aa) (Relative to SEQ IDNO: 6138) RebA^(a) RebM^(a) 6261/6262 V14I/E330D ++ +++ 6263/6264E113S/D158P/L163A/K190R/L257Q/L437I ++ +++ 6265/6266D158P/V320I/L437I/S449R ++ ++ 6267/6268V14I/D158P/L163A/K190R/E342W/L437I + +++ 6269/6270V14I/K190R/L257Q/S317R/V320I/P322L + ++ 6271/6272V14I/E113S/D158P/L163A/K190R/L257Q/R308L/E342W ++ +++ 6273/6274E113S/D158P/L163A/K190R/R308L/V320I ++ +++ 6275/6276 K190R/S449R +++ +++6277/6278 V14I/E113S/D158P/L163A ++ +++ 6279/6280V14I/E113S/L163A/K190R/R308L/S317R ++ +++ 6281/6282 V14I/E113S/D158P +++ 6283/6284 E113S/D158P/K190R/V320I/P322L/S449R ++ +++ 6285/6286E113S/L257Q/S317R/P322L/L437I + ++ 6287/6288E113S/D158P/V320I/P322L/L437I +++ +++ 6289/6290E113S/D158P/K190R/L257Q/V320I ++ +++ 6291/6292 P322L/E330D ++ +++6293/6294 S317R/V320I/P322L/E330D ++ +++ 6295/6296 V14I/S449R ++ +++6297/6298 V14I/E113S/D158P/R308L/S317R/E330D/S449R +++ +++ 6299/6300V14I/E113S/D158P/L163A/L437I +++ +++ 6301/6302 D158P/R308L/V320I/L437I+++ +++ 6303/6304 V14I/D158P/L163A/L257Q/R308L/S449R ++ +++ 6305/6306D158P/L163A/R308L + +++ 6307/6308 V14I/L257Q/R308L/V320I/P322L/E330D +++++ 6309/6310 E113S/D158P/L163A/L257Q/R308L/S317R/P322L/L437I/ ++ +++S449R 6311/6312 V14I/L163A/S317R/V320I + +++ 6313/6314V14I/D158P/L257Q/R308L/L437I/S449R +++ +++ 6315/6316L163A/R308L/E330D/L437I/S449R ++ +++ 6317/6318 D158P/L163A ++ +++6319/6320 V14I/D158P/L163A/L257Q + +++ 6321/6322V14I/D158P/S317R/V320I/E330D/L437I +++ +++ 6323/6324V14I/E113S/V320I/L437I/S449R +++ +++ 6325/6326 E113S/L257Q/R308L ++ +++6327/6328 E113S/D158P/L163A/K190R/R308L/S317R/P322L ++ +++ 6329/6330V14I/K190R + +++ 6331/6332 D158P/L163A/K190R/L257Q/R308L/E342W/S449R ++++ 6333/6334 V14I/D158P/L163A/K190R/L257Q/R308L/S317R/E330D/ +++ +++L437I 6335/6336 V14I/L257Q/R308L/P322L/E330D/L437I +++ +++ 6337/6338V14I/E113S/L163A/L257Q/L437I +++ +++ 6339/6340 V14I/S317R + +++6341/6342 V14I/D158P +++ +++ 6343/6344V14I/E113S/L163A/R308L/S317R/V320I/P322L/E330D/ ++ +++ S449R 6345/6346V14I/E113S/L163A/L257Q/R308L/E342W/L437I/S449R +++ +++ 6347/6348D158P/L163A/P322L/L437I +++ +++ 6349/6350 L29M/D375P + ++ 6351/6352 G84H++ + 6353/6354 R88T + + 6355/6356 D375A + + 6357/6358 Q197K + +6359/6360 I202H + ++ 6361/6362 P83S + + 6363/6364 I202T + + 6365/6366G84N ++ + 6367/6368 A85L + + 6369/6370 V19Q + + 6371/6372 A199M + +6373/6374 I202Q + ++ 6375/6376 N383V + ++ 6377/6378 H81T + + 6379/6380D375V + + 6381/6382 A199Y + + 6383/6384 D72S + + 6385/6386 D375P +++ +++6387/6388 D72T + + 6389/6390 P83N + + 6391/6392 R88K + + 6393/6394 V155L++ ++ 6395/6396 A199H + + 6397/6398 T80P + + 6399/6400 Q71V + +6401/6402 P83A + + 6403/6404 E209T + + 6405/6406 A199E + + 6407/6408P83T + + 6409/6410 I202V + + 6411/6412 G84D + + 6413/6414 R88C + +6415/6416 A199Q +++ ++ 6417/6418 D375T + ++ 6419/6420 R88A + + 6421/6422P83K + + 6423/6424 R88H + + 6425/6426 S41A + ND 6427/6428 A366T + ND6429/6430 A366V ++ ND 6431/6432 E105A + ND 6433/6434 S273R ++ ND6435/6436 S273H ++ ND 6437/6438 V263T + ND 6439/6440 P168C + ND6441/6442 L243I +++ ND 6443/6444 H95N + ND 6445/6446 A366S + ND6447/6448 H46S + ND 6449/6450 S273A + ND 6451/6452 A366L ++ ND 6453/6454A366C +++ ND 6455/6456 Y249S + ND 6457/6458 F45L + ND 6459/6460 P168T +ND aLevels of increased production were determined relative to thereference polypeptide of SEQ ID NO: 6138, and defined as follows: “−” =production less than 0.9-fold; “+” = production at least 0.9-fold, butless than 1.5-fold; “++” = at least 1.5-fold, but less than 1.9-fold;and “+++” = at least 1.9-fold increased production, as compared to thereference polypeptide. ND = not determined.SFP Characterization Assay and Analysis for Glucosyl Transfer fromSucrose to ADP and then from ADP-Glucose to Stevioside and RebaudiosideD

Shake flask powders (SFP) were reconstituted to a concentration of 20g/L and diluted to 0.006-0.2 g/L SFP in 100 μL total reaction volumes of50 mM potassium phosphate buffer, pH6, 10 mM stevioside (>95% purity) orrebaudioside D, 0.2 mM ADP (Sigma, >93% purity) co-substrate, 0.1 g/LSUS SFP SEQ ID NO: 1804, and 24 mM sucrose (cane sugar). The reactionswere incubated at 55° C. with no pre-incubation or at 60° C. following15 minute pre-incubation at 68.6° C. in a Thermotron® titre-plate shakerwith 300 RPM shaking for 4 hours. The reactions were solubilized by 20×dilution in water, quenched by 5× dilution in acetonitrile with 0.2%formic acid, cleared by centrifugation, and diluted 10× in water foranalysis. Glycosylated products were detected in by SPE-QQQ as describedin Example 31, Table 31.1.

TABLE 70.2 β1, 3GT Round 18 Shake Flask Powder Variants and RebA andRebM Levels Increased Increased SEQ ID NO: Amino Acid Differences RebA,RebM, (nt/aa) (Relative to SEQ ID NO: 6138) 60° C^(a) 60° C^(a)6261/6262 V14I/E330D + + 6267/6268 V14I/D158P/L163A/K190R/E342W/ + +++L437I 6287/6288 E113S/D158P/V320I/P322L/L437I +++ +++ 6299/6300V14I/E113S/D158P/L163A/L437I ++ ++ 6333/6334V14I/D158P/L163A/K190R/L257Q/ ++ ++ R308L/S317R/E330D/L437I ^(a)Levelsof increased production were determined relative to the referencepolypeptide of SEQ ID NO: 6138, at 0.025 g/L shake flask powder anddefined as follows: “−” = production less than 1.08-fold; “+” =production at least 1.08-fold, but less than 1.5-fold; “++” = at least1.5-fold, but less than 1.7-fold; and ”+++” = at least 1.7-foldincreased production, as compared to the reference polypeptide.

In these experiments, the five variants in Table 70.2 (SEQ ID NOS: 6262,6268, 6288, 6300, and 6334) produced more rebaudioside A from steviosideand more rebaudioside M from rebaudioside D at 60° C. withpre-incubation than SEQ ID NO: 6138. SEQ ID NO: 6288 had the greatestimprovement for conversion of stevioside to rebaudioside A under theconditions with pre-incubation followed by the 60° C. assay, and was thesecond most improved for rebaudioside D to M, so this variant wasselected for further directed evolution.

Example 71 Beta-1,3-ADP-Glycosyltransferase Variants of SEQ ID NO: 6288

In this Example, experiments for evolution and screening ofβ1,3-glycosyltransferase (β1,3GT) polypeptides derived from SEQ ID NO:6288 for improved glucosylation of steviol glycosides using in situsynthesized ADP-glucose are described. Directed evolution of the GTencoded by SEQ ID NO: 6287 (i.e., SEQ ID NO: 6288) was carried out byconstructing libraries of variant genes in which mutations associatedwith improved production identified during the development of thepresent invention were recombined and in which certain structuralfeatures were subjected to saturation mutagenesis. These libraries werethen plated, grown, and screened using the high-throughput (HTP) assaydescribed below to provide an nineteenth round (“Round 19”) of 108engineered GT variant polypeptides with glucosyltransferase activitytoward ADP-glucose and steviol glycosides.

HTP Assay for Glucose Transfer from Sucrose to ADP and then fromADP-Glucose to Stevioside and Rebaudioside D

Assays were performed on 96-well plates of cleared E. coli culturelysates expressing SEQ ID NO: 6288 variants. Pellets were lysed, andlysate was cleared as described in Example 60 and then diluted 40× into50 mM potassium phosphate buffer, pH 6. In order to thermally challengethe lysates, they were pre-incubated either in a thermocycler for 15minutes at 73.5° C. or at 65° C. in a Thermotron® titre-plate shakerwith 300 RPM shaking for 16 h. Assays were conducted with 10 μLpre-incubated lysate in 100 μL reactions and with 10 mM stevioside (>95%purity) or rebaudioside D substrate, 0.2 mM ADP (Sigma, >93% purity)co-substrate, 0.05 g/L SUS SFP SEQ ID NO: 2064, and 24 mM sucrose (canesugar). The following reaction conditions were used: 50 mM potassiumphosphate buffer, pH 6, 60° C. in a Thermotron® titre-plate shaker with300 RPM shaking for 4 h. Reactions were solubilized by 20× dilution inwater, quenched by 5× dilution in acetonitrile with 0.2% formic acid,and diluted 10× with water for analysis. Samples were analyzed byRapidFire-MS/MS as described in Example 31, Table 31.1.Glycosyltransferase variant polypeptides that produced rebaudiosideA/rebaudioside M from stevioside/rebaudioside D with in situ synthesizedADP-glucose at greater quantities than SEQ ID NO: 6288 were identified.The engineered polypeptides from combinatorial libraries screened withthe 15 minute pre-incubation are listed in Table 71.1, and those fromsaturation mutagenesis libraries screened with the 16 h pre-incubationare listed in Table 71.2. Shake-flask scale cultures were grown, lysed,cleared, and lyophilized to powder as described in Example 1 foranalysis of variants shown in Table 71.3 relative to SEQ ID NO: 6288.

TABLE 71.1 β1, 3GT Round 19 Combinatorial Variants and RebA and RebMLevels SEQ ID NO: Amino Acid Differences Increased Increased (nt/aa)(Relative to SEQ ID NO: 6288) RebA^(a) RebM^(a) 6461/6462S37R/Q71L/K247C/R331K/V365I/D389E/L401F/ + +++ L429W 6463/6464P139K/S156C/K247L/D389E/L401F/A427R/A433L/ + ++++ S449R 6465/6466K247L/H248W/S449R + ++++ 6467/6468P139K/S156C/L174P/D389E/L401F/A427R/A433L/ ++ ++++ S449R 6469/6470P139K/K247C/H248W/R331K/L401F/P417Q/K432L/ + ++++ S449R 6471/6472L174P/D389E/L429W/K432L/A433L/S449R + ++++ 6473/6474 D416S/K432L/A433L +++++ 6475/6476 S37R/H248W/D389A/L401F/L429W + ++++ 6477/6478Q71L/H248W/V365I/D389A/L401F + ++++ 6479/6480P139K/S156C/H248W/D389A/L401F/D416S/A427R/ + ++++ L429W/A433L 6481/6482Q71L/P139K/D389A/A427R/L429W/K432L/A433L/ + +++ S449R 6483/6484S37R/Q71L/L125M/L174P/K247C/H248W/A427R + ++++ 6485/6486S37R/L174P/L401F/E402L/A433L/S449R + ++++ 6487/6488S156C/D389E/L401F/E402L/D416L/K432L/A433L ++ ++++ 6489/6490S254Q/A427R/A433L + +++ 6491/6492S156C/S254Q/R331K/V365I/A427R/K432L/S449R ++ +++ 6493/6494A427R/K432L/S449R +++ ++++ 6495/6496S37R/P139K/A427R/K432L/A433L/S449R + +++ 6497/6498P139K/H248W/E402L/D416L/A427R/A433L/S449R + +++ 6499/6500D416L/K432L/A433L/S449R + ++++ 6501/6502 K247C/A427R/K432L + ++++6503/6504 H248W/L401F/L429W/K432L/A433L + ++++ 6505/6506H248W/R331K/A427R/L429W/A433L ++ +++ 6507/6508 A427R/K432L +++ +++6509/6510 P139K/S254Q/L401F/D416S/A427R/A433L/S449R/ ++ +++ 6511/6512L174P/G329P/K432L/S449R ++ +++ 6513/6514 P139K/L401F/5449R ++ +++6515/6516 P139K/L174P/S254Q ++ +++ 6517/6518P139K/L174P/V365I/L401F/E402L/A427R/A433L/ +++ ++++ S449R 6519/6520D389E/L401F/A427R/K432L +++ +++ 6521/6522 Q71L/A427R/K432L/A433L/S449R +++++ 6523/6524 S37R/P139K/G329P/V365I/A427R/A433L/S449R ++ ++++6525/6526 Q71L/L174P/G329P/A427R/L429W/K432L/A433L + ++++ 6527/6528R331K/V365I/L429W/K432L/A433L/S449R ++ ++++ 6529/6530D416L/A427R/A433L/S449R + ++++ 6531/6532S37R/H248W/R331K/D389E/A427R/A433L + ++++ 6533/6534S37R/P139K/L429W/K432L/A433L ++ ++++ 6535/6536 S156C/H248W/L256M + ++++6537/6538 K247C/H248W/L401F ++ ++++ 6539/6540 S37R/R331K/K432L/A433L ++++++ 6541/6542 K247C/R331K/L401F/A427R/K432L/S449R ++ ++++ 6543/6544D389A/L401F/D416S/K432L/S449R ++ +++ 6545/6546P139K/L174P/H248W/R331K/D389A/L401F/S449R ++ ++++ 6547/6548L174P/K247C/H248W/D389A/L401F/K432L/A433L + +++ 6549/6550 S254Q/V365I +++++ 6551/6552 P139K/S156C/V365I + +++ 6553/6554P139K/S156C/K247C/V365I/L401F/A433L/S449R + ++++ 6555/6556Q71L/H248W/S449R ++ +++ 6557/6558 Q71L/K432L/A433L ++ +++ 6559/6560H248W/D416L ++ +++ 6561/6562 Q71L/S254Q/A433L/S449R ++ +++ 6563/6564A433L/S449R ++ +++ 6565/6566 K247L/L401F ++ +++ 6567/6568H248W/V365I/D389E/A427R/L429W/K432L/5449R ++ +++ 6569/6570Q71L/L174P/V365I/A427R/K432L + +++ 6571/6572 P139K/S156C/H248W/D389E +++++ 6573/6574 P139K/H248W/S254A/5449R ++ +++ 6575/6576 L174P/V365I +++++ 6577/6578 H248W/S449R ++ +++ 6579/6580 H248W/D416L/S449R/ ++ +++6581/6582 K432L + +++ 6583/6584 P139K/H248W/K432L/A433L ++ ++++6585/6586 V365I/L401F/E402L/L429W/K432L/A433L + +++ 6587/6588L401F/A427R ++ +++ ^(a)Levels of increased production were determinedrelative to the reference polypeptide of SEQ ID NO: 6288, and defined asfollows: “−” = production less than that of the reference polypeptide;“+” = production at least that of the reference polypeptide, but lessthan 1.9-fold; “++” = at least 1.9-fold, but less than 2.5-fold; “+++” =at least 2.5-fold, but less than 7-fold; and “++++” = at least 7-foldincreased production, relative to reference polypeptide.

TABLE 71.2 β1, 3GT Round 19 Saturation Mutagenesis Variants and RebA andRebM Levels Amino Acid SEQ ID NO: Differences (Relative IncreasedIncreased (nt/aa) to SEQ ID NO: 6288) RebA^(a) RebM^(a) 6589/6590 M87R +++ 6591/6592 M144Q/S449R +++ +++ 6593/6594 I91Q + + 6595/6596 I91N/S449R− ++ 6597/6598 I91T/S449R + + 6599/6600 L25M/S449R + ++ 6601/6602D69M/S449R + + 6603/6604 M87Q ++ ++ 6605/6606 M87K ++ +++ 6607/6608T361C +++ +++ 6609/6610 L25I/S449R + + 6611/6612 M87A/S449R + +++6613/6614 M87E ++ +++ 6615/6616 A153V + + 6617/6618 A153T/S449R +++ +6619/6620 L25Q/S449R + ++ 6621/6622 L25S + ++ 6623/6624 D69A + +6625/6626 I91V + ++ 6627/6628 I91L + + 6629/6630 W233M/S449R ND +++6631/6632 S317Y ND +++ 6633/6634 W233V ND + 6635/6636 W233S ND +++6637/6638 A212L/S449R ND + 6639/6640 W233L/S449R ND ++ 6641/6642 V369KND + 6643/6644 V421I ND + 6645/6646 W233C ND ++ 6647/6648 W233Q/S449R ND+++ 6649/6650 E6P ND − 6651/6652 K347P/S449R ND + 6653/6654 Q159R/S449RND ++ 6655/6656 Q159K ND +++ 6657/6658 E288P/S449R ND ++ 6659/6660 H172SND + 6661/6662 T5S/S449R ND + 6663/6664 W233G ND + 6665/6666 Q303C ND +6667/6668 W233A ND ++ 6669/6670 R10K ND + 6671/6672 W233R ND ++6673/6674 H172T/S449R ND + 6675/6676 Q303V ND + ^(a)Levels of increasedproduction were determined relative to the reference polypeptide of SEQID NO: 6288, and defined as follows: “−” = production less than that ofthe reference polypeptide; “+” = production at least that of thereference polypeptide, but less than 1.28-fold; “++” = at least1.28-fold, but less than 1.5-fold; and “+++” = at least 1.5-foldincreased production, relative to the reference polypeptide.SFP Characterization Assay and Analysis for Glucosyl Transfer fromSucrose to ADP and then from ADP-Glucose to Stevioside and RebaudiosideD

Shake flask powders (SFP) were reconstituted to a concentration of 20μLand diluted to 0.006-0.2 g/L SFP in 100 μL total reaction volumes of50 mM potassium phosphate buffer, pH6, 10 mM stevioside (>95% purity) orrebaudioside D, 0.2 mM ADP (Sigma, >93% purity) co-substrate, 0.05 g/LSUS SFP SEQ ID NO: 2064, and 24 mM sucrose (cane sugar). The reactionswere incubated in a Thermotron® titre-plate shaker with 300 RPM shakingfor 4 hours at 55° C. with no pre-incubation or at 60° C. following 15minute pre-incubation at 73.5° C. The reactions were solubilized by 20×dilution in water, quenched by 5× dilution in acetonitrile with 0.2%formic acid, cleared by centrifugation, and diluted 10× with water foranalysis. Glycosylated products were detected in by SPE-QQQ as describedin Example 31, Table 31.1. A further thermostability characterizationwas conducted with the clarified shake flask lysates prior tolyophilization, as follows: lysates were diluted 400× in buffer andincubated in a thermocycler at a gradient of 55-70° C. for 16 h. Todetermine the percent of remaining activity, the pre-incubated lysateswere assayed as described above, with either stevioside or rebaudiosideD and 4h incubation at 60′C. The percent activity remaining is expressedas production at the high temperature divided by production at lowestpre-incubated temperature.

TABLE 71.3 β1, 3GT Round 19 Shake Flask Powder Variants and RebA, RebM,and Thermostability Levels SEQ ID Amino Acid Differences IncreasedIncreased Increased NO: (Relative to RebA, RebA, RebM, % Activity(nt/aa) SEQ ID NO: 6288) 55° C^(a) 60° C^(a) 60° C^(a) Remaining^(b)6467/6468 P139K/S156C/L174P/D389E/ + ++ +++ +++ L401F/A427R/A433L/S449R6493/6494 A427R/K432L/S449R + ++ ++ ++ 6517/6518P139K/L174P/V365I/L401F/ − + ++ +++ E402L/A427R/A433L/S449R 6519/6520D389E/L401F/A427R/K432L + ++ ++ +++ 6527/6528 R331K/V365I/L429W/K432L/− + +++ + A433L/S449R 6539/6540 S37R/R331K/K432L/A433L + + +++ −6573/6574 P139K/H248W/S254A/S449R − + +++ + 6583/6584P139K/H248W/K432L/A433L − + +++ ++ ^(a)Levels of increased productionwere determined relative to the reference polypeptide of SEQ ID NO:6288, at 0.025 g/L shake flask powder and defined as follows: “−” =production less than 0.9-fold; “+” = production at least 0.9-fold, butless than 3.2-fold; “++” = at least 3.2-fold, but less than 5.5-fold;and “+++” = at least 5.5-fold increased production, relative toreference polypeptide. ^(b)The percent levels of activity remaining weredetermined following 16 h pre-incubation at 70° C. relative to eachvariant following 16 h pre-incubation at 55° C. and defined as follows:“−” = less than 20% of activity remained following 16 h pre-incubationat 55° C.; “+” = at least 20%, but less than 40% of the activityremained; “++” = at least 40%, but less than 60% of the activityremained; and “+++” = at least 60% of the activity remained.

In these experiments, the eight variants in Table 71.3 produced morerebaudioside A from stevioside and more rebaudioside M from rebaudiosideD at 60° C. with pre-incubation than SEQ ID NO: 6288. SEQ ID NO: 6468had the greatest improvement for conversion of rebaudioside D torebaudioside M under the conditions with pre-incubation followed by the60° C. assay and had the greatest percent activity remaining following16 h thermochallenge, so this variant was selected for further directedevolution.

Example 72 Beta-1,3-ADP-Glycosyltransferase Variants of SEQ ID NO: 6468

In this Example, experiments for evolution and screening ofβ1,3-glycosyltransferase (β1,3GT) polypeptides derived from SEQ ID NO:6468 for improved glucosylation of steviol glycosides using in situsynthesized ADP-glucose are described. Directed evolution of the GTencoded by SEQ ID NO: 6467 (i.e., SEQ ID NO: 6468) was carried out byconstructing libraries of variant genes in which mutations associatedwith improved production identified during the development of thepresent invention were recombined and in which certain structuralfeatures were subjected to saturation mutagenesis. These libraries werethen plated, grown, and screened using the high-throughput (HTP) assaydescribed below to provide a twentieth round (“Round 20”) of 269engineered GT variant polypeptides with glucosyltransferase activitytoward ADP-glucose and steviol glycosides.

HTP Assay for Glucose Transfer from Sucrose to ADP and then fromADP-Glucose to Stevioside and Rebaudioside D

Assays were performed on 96-well plates of cleared E. coli culturelysates expressing SEQ ID NO: 6467 variants. Pellets were lysed, andlysate was cleared as described in Example 60, and then diluted 40× into50 mM potassium phosphate buffer, pH 6. In order to thermally challengethe lysates, they were pre-incubated either in a thermocycler for 15minutes at 79° C. or at 65° C. in a Thermotron® titre-plate shaker with300 RPM shaking for 16 h. Assays were conducted with 10 μL pre-incubatedlysate in 100 μL reactions and with 15 mM stevioside (>95% purity) orrebaudioside D substrate, 0.1 g/L ADP (Sigma, >93% purity) co-substrate,0.05 g/L SUS SFP SEQ ID NO: 2432, and 37.5 mM sucrose (cane sugar). Thefollowing reaction conditions were used: 50 mM potassium phosphatebuffer, pH 6, 60° C. in a Thermotron® titre-plate shaker with 300 RPMshaking for 4 h. Reactions were solubilized by 20× dilution with water,quenched by 5× dilution in acetonitrile with 0.2% formic acid, anddiluted 15× with water for analysis. Samples were analyzed byRapidFire-MS/MS as described in Example 31, Table 31.1.Glycosyltransferase variant polypeptides that produced rebaudiosideA/rebaudioside M from stevioside/rebaudioside D with in situ synthesizedADP-glucose at greater quantities than SEQ ID NO: 6468 were identified.The engineered polypeptides from combinatorial libraries screened withthe 15 minute pre-incubation are listed in Table 72.1, and those fromsaturation mutagenesis libraries screened with the 16 h pre-incubationare listed in Table 72.2. Shake-flask scale cultures were grown, lysed,cleared, and lyophilized to powder as described in Example 31 foranalysis of variants shown in Table 72.3 relative to SEQ ID NO: 6468.

TABLE 72.1 β1, 3GT Round 20 Combinatorial Variants and RebA and RebMLevels SEQ ID NO: Amino Acid Differences Increased Increased (nt/aa)(Relative to SEQ ID NO: 6468) RebA^(a) RebM^(a) 6677/6678D72T/P83S/G84N/R88T/I202H/S254A/S273R/A366C/ ++ ++ D375P 6679/6680F45L/D72T/R88T/A366C ++ ++ 6681/6682 P83S/R88T/V155L/S273R/A366V/D375P++ +++ 6683/6684 H81T/R88T/D375P/E402L ++ +++ 6685/6686S37R/D72T/Q197K/S273R/R331K/D375P/K432L ++ +++ 6687/6688D72T/V155L/K190R ++ +++ 6689/6690 H248W/D375P ++ +++ 6691/6692D72T/H248W ++ +++ 6693/6694 H81T/P83S/I202H/V365I/A366C/E402L + +++6695/6696 A199Q/V263T/R331K/V365I/A366C ++ ++ 6697/6698 V365I/A366C ++++ 6699/6700 F45L/D72T/P168T/L243I/R331K/V365I/A366C/L429W/ ++ ++ K432L6701/6702 D72T/P83S/R88T/L243I/V263T/R331K/V365I/A366C ++ ++ 6703/6704H81T/P83S/K190R/V263T/V365I/A366C ++ +++ 6705/6706S37R/D72T/H81T/P83S/R88T/V155L/K190R ++ ++ 6707/6708F45L/D72T/G84N/R88T/Q197K/D375P ++ ++++ 6709/6710G84N/Q197K/A366C/E402L + +++ 6711/6712F45L/L163A/P168T/K190R/A199Q/A366C/L429W/ ++ ++ K432L 6713/6714G84N/V155L/P168T/Q197K/A199Q/R331K/A366V/ ++ + D375P/N383V/E402L6715/6716 K190R/A199Q/I202H/R331K/A366C + ++++ 6717/6718S37R/K190R/I202H + +++ 6719/6720D72T/P83S/G84N/Q197K/I202H/L243I/V263T/V365I/ ++ +++ A366C 6721/6722Q197K/A199Q/I202H − +++ 6723/6724 G84N/P168T/Q197K/I202H/V263T/A366C ++++ 6725/6726 Q197K/I202H/H248W + ++++ 6727/6728 Q197K/H248W ++ ++++6729/6730 S37R/P83S/V263T/V365I/A366V/D375P ++ +++ 6731/6732F45L/L163A/P168T/Q197K/V263T/R331K/V3651/A366C + +++ 6733/6734F45L/D72T/L163A/I202H/V365I/A366V/D375P ++ ++++ 6735/6736D72T/H81T/G84N/K190R/H248W ++ +++ 6737/6738 H81T/P83S/E169D/K190R/V263T++ +++ 6739/6740 F45L/G84N/P168T/K190R/A199Q/S254A/S273R/V365I/ ++ +A366C 6741/6742 V365I/D375P/E402L + +++ 6743/6744V155L/P168T/K190R/Q197K/A199Q/A366C ++ +++ 6745/6746S37R/S41A/F45L/D72T/V155L/K190R/L2431/H248W/ + +++S273R/R331K/K432L/L433A 6747/6748S37R/S41A/F45L/D72T/V155L/L163A/R331K/A366C/ ++ +++ D375P 6749/6750D72T/H81T/P83S/G84N/R88T/V155L/K190R ++ ++ 6751/6752V155L/V263T/A366C/K432L/L433A ++ +++ 6753/6754S37R/S41A/F45L/D72T/V155L/V263T/D375P ++ +++ 6755/6756S37R/S41A/F45L/V155L/A366C/K432L/L433A ++ +++ 6757/6758D72T/H81T/P83S/G84N/R88T/L163A/P168T/V263T/ ++ ++ R331K/D375P 6759/6760S37R/S41A/F45L/V155L/A366C ++ ++ 6761/6762S37R/S41A/F45L/D72T/V155L/L163A/P168T/L243I/ + +++H248W/S273R/R331K/A366V/K432L/L433A 6763/6764S37R/S41A/F45L/D72T/V155L/K190R/S273R/R331K/ ++ ++ A366V/D375P 6765/6766S37R/S41A/F45L/L243I/H248W/S273R/R331K + ++ 6767/6768S37R/S41A/F45L/H81T/P83S/G84N/R88T/L163A/P168T/ ++ ++V263T/S273R/R331K/A366C/D375P 6769/6770S37R/S41A/F45L/D72T/V155L/K190R/S273R/A366C ++ +++ 6771/6772S37R/S41A/F45L/V155L/P168T/H248W/S273R/R331K/ + ++ D375P 6773/6774S37R/S41A/F45L/V155L/L163A/P168T/V263T/R331K/ ++ +++ D375P 6775/6776S37R/S41A/F45L/D72T/V155L/R331K/A366V/D375P/ ++ +++ K432L/L433A6777/6778 S37R/S41A/F45L/V155L/K190R/R331K/A366V/D375P ++ +++ 6779/6780H81T/P83S/G84N/R88T/V155L/L163A/P168T/S273R/ ++ ++ R331K/D375P 6781/6782D72T/H81T/P83S/G84N/R88T/L163A/P168T/K190R/ ++ ++L243I/V263T/R331K/A366C 6783/6784S37R/S41A/F45L/D72T/V155L/H248W/V263T/S273R/ ++ +++ A366C 6785/6786V155L/P168T/D375P ++ +++ 6787/6788S41A/F45L/L163A/P168T/L243I/H248W/S273R/A366C/ + +++ K432L 6789/6790S37R/S41A/F45L/V155L/D375P ++ +++ 6791/6792D72T/L243I/H248W/A366V/K432L/L433A + +++ 6793/6794D72T/L2431/H248W/S273R/A366V/K432L/L433A + +++ 6795/6796S37R/S41A/F45L/D72T/V155L/V263T/R331K/D375P ++ +++ 6797/6798D72T/V155L/S273R/R331K/D375P/K432L ++ +++ 6799/6800D72T/L243I/H248W/V263T/A366V/K432L/L433A ++ +++ 6801/6802S37R/S41A/F45L/D72T/H81T/P83S/G84N/R88T/V155L/ ++ ++ R331K/A366C/D375P6803/6804 S37R/S41A/F45L/H81T/P83S/G84N/R88T/V155L/P168T/ ++ ++R331K/D375P 6805/6806 S37R/S41A/F45L/D72T/H81T/P83S/G84N/R88T/V155L/ ++++ R331K/D375P 6807/6808 S37R/S41A/F45L/D72T/V155L/K190R/H248W/V263T/ +++++ R331K 6809/6810 S37R/S41A/F45L/D72T/V263T/A366C/K432L/L433A ++ +++6811/6812 S37R/S41A/F45L/D72T/V155L/S273R/A366C ++ ++ 6813/6814S37R/S41A/F45L/D72T/H81T/P83S/G84N/R88T/V155L/ ++ ++V263T/S273R/R331K/D375P/K432L 6815/6816S37R/S41A/F45L/V263T/R331K/D375P/K432L ++ +++ 6817/6818S37R/S41A/F45L/D72T/R331K/A366C ++ ++ 6819/6820H81T/P83S/G84N/R88T/V263T/S273R/R331K/A366C/K ++ ++ 432L 6821/6822H81T/P83S/G84N/R88T/V155L/A366C ++ ++ 6823/6824S37R/S41A/F45L/D72T/R88T/V155L/P168T/K190R/ ++ ++R331K/A366C/D375P/K432L 6825/6826S37R/S41A/F45L/D72T/V155L/L163A/P168T/V263T/ ++ ++ A366C/K432L/L433A6827/6828 H81T/P83S/G84N/R88T/V155L/V263T/A366V/D375P ++ ++ 6829/6830S37R/S41A/F45L/D72T/V155L/L163A/P168T/K190R/ ++ ++L2431/V263T/S273R/R331K/A366C/K432L 6831/6832S37R/S41A/F45L/D72T/H81T/P83S/G84N/R88T/H248W/ ND + D375P 6833/6834D72T/H81T/P83S/G84N/R88T/V155L/L163A/P168T/ ++ + K190R/A366C 6835/6836S37R/S41A/F45L/D72T/V155L/K190R/V263T/R331K/ ++ ++ A366C 6837/6838D72T/V155L/K190R/A366C ++ ++ 6839/6840S37R/S41A/F45L/D72T/K190R/V263T/S273R/R331K ND ++ 6841/6842D72T/V155L/K190R/V263T/R331K/A366C ++ ++ 6843/6844S37R/S41A/F45L/D72T/K190R/A366C ++ ++ 6845/6846H81T/P83S/G84N/R88T/K190R/V263T/D375P ++ ++ 6847/6848D72T/H81T/P83S/G84N/R88T/V155L/A366C ++ ++ 6849/6850D72T/H81T/P83S/G84N/R88T/V155L/S273R/R331K/ ++ ++ D375P 6851/6852S37R/S41A/F45L/R331K/A366V/K432L/L433A ++ + 6853/6854S37R/S41A/F45L/D72T/R88T/V155L/P168T/K190R/ ++ ++ R331K/D375P 6855/6856S37R/S41A/F45L/D72T/H81T/P83S/G84N/R88T/V155L/ ++ ++L163A/P168T/K190R/R331K/D375P 6857/6858D72T/H81T/P83S/G84N/R88T/V155L/K190R/S273R/ ++ ++ R331K/A366V/K432L6859/6860 S41A/F45L/D72T/V155LN263T/R331K/A366V/D375P/ ++ ++ K432L/L433A6861/6862 S37R/S41A/F45L/D72T/H81T/P83S/G84N/R88T/R331K/ ++ ++A366V/K432L/L433A 6863/6864 S37R/S41A/F45L/D72T/K190R/R331K/A366C ++ ++6865/6866 S37R/S41A/F45L/H81T/P83S/G84N/R88T/V155L/P168T/ ++ +K190R/L243I/R331K/A366C 6867/6868S37R/S41A/F45L/H81T/P83S/G84N/R88T/V155L/V263T/ ++ ++ S273R/R331K/A366C6869/6870 S37R/S41A/F45L/D72T/R88T/K190R/V263T/S273R/ ++ ++ R331K/A366C6871/6872 S37R/S41A/F45L/D72T/V263T/R331K/A366C ++ ++ 6873/6874H81T/P83S/G84N/R88T/V155L/K190R/V263T/R331K/ ++ ++ A366C 6875/6876S37R/S41A/F45L/D72T/V155L/K190R/S273R/A366C/ ++ ++ D375P/K432L 6877/6878D72T/V155L/L163A/P168T/K190R/A366V/K432L/L433A ++ ++ ^(a)Levels ofincreased production were determined relative to the referencepolypeptide of SEQ ID NO: 6468, and defined as follows: “−” = productionat least that of the reference polypeptide; “+” = at least that of thereference polypeptide, but less than 2-fold; “++” = at least 2-fold, butless than 4-fold; “+++” = at least 4-fold, but less than 8-fold; and“++++” = at least 8-fold increased production, as compared to thereference polypeptide. ND = not determined.

TABLE 72.2 β1, 3GT Round 20 Saturation Mutagenesis Variants and RebALevels Amino Acid SEQ ID NO: Differences (Relative to (nt/aa) SEQ ID NO:6468) Increased RebA^(a) 6879/6880 L322K +++ 6881/6882 R435S +++6883/6884 V9K + 6885/6886 P439G + 6887/6888 Q265H + 6889/6890 S253D +6891/6892 S61Q +++ 6893/6894 P2Q + 6895/6896 P2H + 6897/6898 N408S +6899/6900 P439A ++ 6901/6902 K428R ++ 6903/6904 D170A + 6905/6906 G96K +6907/6908 D98E + 6909/6910 K171Q + 6911/6912 R296Q + 6913/6914 A118V ++6915/6916 L229M + 6917/6918 P2K +++ 6919/6920 S253P +++ 6921/6922 K222Q++ 6923/6924 P439H ++ 6925/6926 A118T/L120V ++ 6927/6928 T4N + 6929/6930M183L ++ 6931/6932 Q269N ++ 6933/6934 S253T +++ 6935/6936 P272S +6937/6938 R435Y ++ 6939/6940 K289N + 6941/6942 S253V + 6943/6944 K222A++ 6945/6946 A438S ++ 6947/6948 R435G ++ 6949/6950 S442T + 6951/6952R435D ++ 6953/6954 L322P/V407I + 6955/6956 E226S + 6957/6958 D170V +6959/6960 P2R + 6961/6962 L322V/V407I +++ 6963/6964 R173K ++ 6965/6966N399S + 6967/6968 N3M ++ 6969/6970 M183I +++ 6971/6972 K428L + 6973/6974K302G + 6975/6976 K222R + 6977/6978 R435N ++ 6979/6980 Q269R +++6981/6982 R435E +++ 6983/6984 V9S ++ 6985/6986 S442F +++ 6987/6988 Y396T++ 6989/6990 K428I +++ 6991/6992 S214R ++ 6993/6994 E405P + 6995/6996R234S + 6997/6998 P439E + 6999/7000 N3F + 7001/7002 V390I +++ 7003/7004P439R + 7005/7006 Y193F ++ 7007/7008 R403V ++ 7009/7010 K171L +7011/7012 P158T + 7013/7014 N408D + 7015/7016 G96C + 7017/7018 K171A ++7019/7020 Q269M + 7021/7022 D300E + 7023/7024 K222N + 7025/7026 D170G +7027/7028 P2M + 7029/7030 K428Y + 7031/7032 K94R + 7033/7034 L322T ++7035/7036 E448Q + 7037/7038 D98T ++ 7039/7040 N3L + 7041/7042 R411K ++7043/7044 D170H + 7045/7046 Q269L + 7047/7048 K171P +++ 7049/7050 E405S++ 7051/7052 M183P ++ 7053/7054 V395I/P439V +++ 7055/7056 L322S +++7057/7058 V9A + 7059/7060 R449L + 7061/7062 Y165L +++ 7063/7064 A438E +7065/7066 R234T ++ 7067/7068 L229Q + 7069/7070 K428G ++ 7071/7072 N399P+++ 7073/7074 F64M + 7075/7076 E330S ++ 7077/7078 N408K + 7079/7080D434G + 7081/7082 E405T + 7083/7084 N399Q ++ 7085/7086 R435L ++7087/7088 R435V + 7089/7090 P2F ++ 7091/7092 E448K + 7093/7094 F64L +7095/7096 K53E/I437T +++ 7097/7098 R411H + 7099/7100 K428V +++ 7101/7102K428T ++ 7103/7104 R173L + 7105/7106 D98S + 7107/7108 P439W + 7109/7110Y396V + 7111/7112 S113P ++ 7113/7114 K428Q +++ 7115/7116 V9M + 7117/7118A438R ++ 7119/7120 D434E +++ 7121/7122 R411T +++ 7123/7124 R234N ++7125/7126 K428S +++ 7127/7128 D300A + 7129/7130 R173S + 7131/7132K289R + 7133/7134 L322P +++ 7135/7136 S113G + 7137/7138 R423T +7139/7140 P439M + 7141/7142 E405A + 7143/7144 S214R/K222H + 7145/7146D72E/D170A ++ 7147/7148 T4S +++ 7149/7150 R449G + 7151/7152 D134E/P158N+++ 7153/7154 R435K + 7155/7156 L322A ++ 7157/7158 S452T + 7159/7160R412K +++ 7161/7162 A118C + 7163/7164 Y444A + 7165/7166 E405D +7167/7168 L120V ++ 7169/7170 S253E +++ 7171/7172 P2V + 7173/7174 D170P++ 7175/7176 K289G + 7177/7178 K428E ++ 7179/7180 V390R + 7181/7182S304P + 7183/7184 S214K ++ 7185/7186 R173I + 7187/7188 S113D +++7189/7190 R435I +++ 7191/7192 D72E/E405S ++ 7193/7194 R435A + 7195/7196E398S + 7197/7198 I454V ++ 7199/7200 R449S + 7201/7202 K428N +++7203/7204 V9G ++ 7205/7206 K289D + 7207/7208 S304K ++ 7209/7210 L322G ++7211/7212 A129P + 7213/7214 S253N + ^(a)Levels of increased productionwere determined relative to the reference polypeptide of SEQ ID NO:6468, and defined as follows: “+” = production at least 1.05-fold, butless than 1.14-fold; “++” = at least 1.14-fold, but less than 1.2-fold;and “+++” = at least 1.2-fold increased production as compared to thereference polypeptide.SFP Characterization Assay and Analysis for Glucosyl Transfer fromSucrose to ADP and then from ADP-Glucose to Stevioside and RebaudiosideD

Shake flask powders (SFP) were reconstituted to a concentration of 20g/L and diluted to 0.006-0.2 g/L SFP in 100 μL total reaction volumes of50 mM potassium phosphate buffer, pH 6, 15 mM stevioside (>95% purity)or rebaudioside D, 0.2 mM ADP (Amresco, ultra pure grade) co-substrate,0.05 g/L SUS SFP SEQ ID NO: 2432, and 37.5 mM sucrose (cane sugar). Thereactions were incubated in a Thermotron® titre-plate shaker with 300RPM shaking for 4 hours at 55° C. with no pre-incubation or at 60° C.following 15 minute pre-incubation at 79° C. The reactions weresolubilized by 20× dilution with water, quenched by 5× dilution inacetonitrile with 0.2% formic acid, cleared by centrifugation, anddiluted 15× with water for analysis. Glycosylated products were detectedin by SPE-QQQ as described in Example 31, Table 31.1. A furtherthermostability characterization was conducted with the clarified shakeflask lysates prior to lyophilization as follows: lysates were diluted400× in buffer and incubated in a thermocycler at a gradient of 60-75°C. for 16 h. To determine the percent activity remaining, thepre-incubated lysates were assayed as described above with eitherstevioside or rebaudioside D and 4 h incubation at 60° C. The percent ofthe remaining activity is expressed as production at the hightemperature divided by production at lowest pre-incubated temperature.

TABLE 72.3 β1, 3GT Round 20 Shake Flask Powder Variants and RebA, RebM,and Thermostability Levels Amino Acid SEQ Differences IncreasedIncreased Increased ID NO: (Relative to RebA, RebA, RebM, % Activity(nt/aa) SEQ ID NO: 6468) 55° C.^(a) 60° C.^(a) 60° C.^(a) Remaining^(b)6739/6740 F45L/G84N/P168T/K190R/ + +++ ++ + A199Q/S254A/S273R/V365I/A366C 6873/6874 H81T/P83S/G84N/R88T/ + +++ + + V155L/K190R/V263T/R331K/A366C 6863/6864 S37R/S41A/F45L/D72T/ + ++ ++ + K190R/R331K/A366C6755/6756 S37R/S41A/F45L/V155L/ + +++ ++ ++ A366C/K432L/L433A 6809/6810S37R/S41A/F45L/D72T/ + +++ ++ ++ V263T/A366C/K432L/L433A 6743/6744V155L/P168T/K190R/ + ++ + + Q197K/A199Q/A366C 6695/6696A199Q/V263T/R331K/ + +++ + ++ V365I/A366C 6837/6838D72T/V155L/K190R/A366C + ++ ++ − 6849/6850 D72T/H81T/P83S/G84N/ + ++ +++ R88T/V155L/S273R/ R331K/D375P 6707/6708 F45L/D72T/G84N/ + +++ + +++R88T/Q197K/D375P 6681/6682 P83S/R88T/V155L/ − +++ ++ ++S273R/A366V/D375P 6697/6698 V365I/A366C − + + + 6785/6786V155L/P168T/D375P − ++ ++ +++ 6725/6726 Q197K/I202H/H248W − + ++ ++^(a)Levels of increased production were determined relative to thereference polypeptide of SEQ ID NO: 6468, at 0.025 g/L shake flaskpowder and defined as follows: “−” = production less than that of thereference polypeptide; “+” = production at least that of the referencepolypeptide, but less than 1.8-fold; “++” = at least 1.8-fold, but lessthan 2.5-fold; and “+++” = at least 2.5-fold increased production,relative to reference polypeptide. ^(b)The percent of activity remainingfor each variant was determined following 16 h pre-incubation at 70° C.,relative to the production of each variant following 16 h pre-incubationat 60° C., and defined as follows: “−” = less than 40% of activityremained following 16 h pre-incubation at 60° C.; “+” = at least 40%,but less than 60% of the activity remained; “++” = at least 60%, butless than 80% of the activity remained; and “+++” = at least 80% of theactivity remained.

In these experiments, the fourteen variants in Table 72.3 produced morerebaudioside A from stevioside and more rebaudioside M from rebaudiosideD at 60° C. with pre-incubation than SEQ ID NO: 6468, and all but fouralso outperformed at 55° C. without pre-incubation. SEQ ID NO: 6864 wasselected as the best enzyme for the catalysis of glycosyltransfer fromADP-glucose to stevioside and rebaudioside D for the formation ofrebaudioside A and rebaudioside M, respectively.

Example 73 Beta-1,3-ADP-Glycosyltransferase Variants of SEQ ID NO: 6864

In this Example, experiments for evolution and screening ofβ1,3-glycosyltransferase (β1,3GT) polypeptides derived from SEQ ID NO:6864 for improved glucosylation of steviol glycosides using in situsynthesized ADP-glucose are described. Directed evolution of the GTencoded by SEQ ID NO: 6863 (i.e., SEQ ID NO: 6864) was carried out byconstructing libraries of variant genes in which mutations associatedwith improved production identified during the development of thepresent invention were recombined. These libraries were then plated,grown, and screened using the high-throughput (HTP) assay describedbelow to provide a twenty-first round (“Round 21”) of 37 engineered GTvariant polypeptides with glucosyltransferase activity towardADP-glucose and steviol glycosides.

HTP Assay for Glucose Transfer from Sucrose to ADP and then fromADP-Glucose to Rebaudioside A 60

Assays were performed on 96-well plates of cleared E. coli culturelysates expressing SEQ ID NO: 6863 variants. Pellets were lysed, andlysate was cleared as described in Example 34 and then diluted 40× into50 mM potassium phosphate buffer, pH 6. In order to thermally challengethe lysates, they were pre-incubated at 75° C. in a Thermotron®titre-plate shaker with 300 RPM shaking for 1h. Assays were conductedwith 10 μL pre-incubated lysate in 100 μL reactions and with 20 g/Lrebaudioside A60% substrate, 0.1 g/L ADP (Amresco, ultra pure grade)co-substrate, 0.05 g/L SUS SFP SEQ ID NO: 2510, 0.1 g/Lβ-1,2-glycosyltransferase/(β12GT) SEQ ID NO:4550 and 37.5 mM sucrose(cane sugar). The following reaction conditions were used: 50 mMpotassium phosphate buffer, pH 6, 60° C. in a Thermotron® titre-plateshaker with 300 RPM shaking for 4h. Reactions were solubilized by 20×dilution with water, quenched by 5× dilution with acetonitrile with 0.2%formic acid, and diluted 15× with water for analysis. Samples wereanalyzed by RapidFire-MS/MS as described in Example 31, Table 31.1.Glycosyltransferase variant polypeptides that produced rebaudiosideA/rebaudioside M from stevioside/rebaudioside D with in situ synthesizedADP-glucose at greater quantities than SEQ ID NO: 6864 were identified.The engineered polypeptides are listed in Table 73.1. Shake-flask scalecultures were grown, lysed, cleared, and lyophilized to powder asdescribed in Example 31 for analysis of variants shown in Table 73.2relative to SEQ ID NO: 6864.

TABLE 73.1 β1, 3GT Round 21 Combinatorial Variants and RebM Levels SEQAmino Acid ID NO: Differences Increased (nt/aa) (Relative to SEQ ID NO:6864) RebM^(a) 7361/7362 R109W/M144Q/A153V/V155L/Q159K/L433A + 7363/7364M87K/M144Q +++ 7365/7366 M87K/M144Q/Q159K/T361C + 7367/7368 M87K/L433A +7371/7372 M144Q +++ 7373/7374 M87K/M144Q/Q159K/T361C/L433A + 7375/7376R88T/M144Q/Q159K +++ 7377/7378 R88T/M144Q ++ 7379/7380T5S/D69A/I91Q/A212L/Q303V ++ 7381/7382 V263T/E288P/Q303V/S317Y ++7383/7384 T5S/E6P/D69A/E288P/Q303V ++ 7385/7386 T5S/L25Q/E288P +7387/7388 T5S/E6P/I91Q/E288P/S317Y +++ 7389/7390 T5S/I91Q/S317Y/V4211 +7391/7392 L25Q/I91Q/V263T/E288P/Q303V + 7393/7394 E288P + 7395/7396T5S/L25Q/I91Q/V263T + 7397/7398 T5S/I91Q/Q303V ++ 7399/7400 T5S/E288P ++7401/7402 E6P/A212L/E288P/Q303V + 7403/7404L25Q/191T/Q303V/S317Y/V369K + 7405/7406 L25Q/V263T/S317Y +++ 7407/7408E6P/I91Q/A212L/E288P/Q303V/V369K/V4211 + 7409/7410I91Q/E288P/S317Y/V369K/V4211 ++ 7411/7412 L25Q/I91Q/A212L/E288P +7413/7414 A212L/E288P + 7415/7416 I91Q/Q303V +++ 7417/7418I91Q/E288P/Q303V/S317Y/V369K ++ 7419/7420 I91Q/S317Y ++ 7421/7422T5S/I91Q/E288P/Q303V +++ 7423/7424 T5S/E6P/L25I/S317Y ++ 7425/7426I91Q/V263T/S317Y/V369K ++ 7427/7428 I91Q/S317Y/V369K + 7429/7430T5S/D69A/I91Q/A212L/E288P + 7431/7432 T5S/L25Q/I91Q/A212L/Q303V/S317Y ++7433/7434 E6P/E288P + 7435/7436 L25Q/I91Q/S317Y/V369K + ^(a)Levels ofincreased production were determined relative to the referencepolypeptide of SEQ ID NO: 6864, and defined as follows: “+” = at least1.12-fold, but less than 1.23-fold; “++” = at least 1.23-fold, but lessthan 1.33-fold; and “+++” = at least 1.33-fold increased production, ascompared to the reference polypeptide.SFP Characterization Assay and Analysis for Glucosyl Transfer fromSucrose to ADP and then from ADP-Glucose to Stevioside and RebaudiosideD

Shake flask powders (SFP) were reconstituted to a concentration of 20g/L and diluted to 0.003-0.1 g/L SFP in 100 μL total reaction volumes of50 mM potassium phosphate buffer, pH6, 15 mM stevioside (>95% purity) orrebaudioside D, 0.2 mM ADP (Amresco, ultra pure grade) co-substrate,0.05 g/L SUS SFP SEQ ID NO: 2510, and 37.5 mM sucrose (cane sugar). Thereactions were incubated in a Thermotron® titre-plate shaker with 300RPM shaking for 4 hours at 60° C. The reactions were solubilized by 20×dilution with water, quenched by 5× dilution with acetonitrile with 0.2%formic acid, cleared by centrifugation, and diluted 15× with water foranalysis. Glycosylated products were detected in by SPE-QQQ as describedin Example 31, Table 31.1. A further thermostability characterizationwas conducted with the clarified shake flask lysates prior tolyophilization as follows: lysates were diluted 400× in 50 mM potassiumphosphate buffer, pH6, and incubated in a thermocycler at a gradient of60-75° C. for 16 h. To determine the percent of activity remaining, thepre-incubated lysates were assayed as described above with eitherstevioside or rebaudioside D and 4h incubation at 60° C. The percent ofactivity remaining is expressed as production at the high temperaturedivided by production at the lowest pre-incubated temperature.

TABLE 73.2 β1, 3GT Round 21 Shake Flask Powder Variants and RebA, RebM,and Thermostability Levels Amino Acid SEQ Differences IncreasedIncreased ID NO: (Relative to SEQ RebM, RebA, % Activity (nt/aa) ID NO:6864) 60° C.^(a) 60° C.^(a) Remaining^(b) 7369/7370 M87K/M144Q + ++ +7375/7376 R88T/M144Q/Q159K + ++ − 7387/7388 T5S/E6P/I91Q/E288P/S317Y++ + + 7405/7406 L25Q/V263T/S317Y + − ++ 7423/7424 T5S/E6P/L25I/S317Y −− + 7431/7432 T5S/L25Q/I91Q/A212L/ − − ++ Q303V/S317Y ^(a)Levels ofincreased production were determined relative to the referencepolypeptide of SEQ ID NO: 6864, at 0.0125 g/L shake flask powder anddefined as follows: “−” = production less than that of the referencepolypeptide; “+” = production at least that of the referencepolypeptide, but less than 1.15-fold; “++” = at least 1.15-foldincreased production, relative to reference polypeptide. ^(b)The percentof activity remaining for each variant was determined following 24 hpre-incubation at 73.5° C., relative to the production from each variantfollowing 24 h pre-incubation at 60° C. and is defined as follows: “−” =less than 20% of activity remained following 16 h pre-incubation at 60°C..; “+” = at least 20%, but less than 40% activity remained; and “++” =at least 40% activity remained.

In these experiments, five variants in Table 73.2 produced morerebaudioside A from stevioside and more rebaudioside M from rebaudiosideD at 60° C. than SEQ ID NO: 6864. SEQ ID NO: 7388 was selected as thebest enzyme for the catalysis of glycosyltransfer from ADP-glucose tostevioside and rebaudioside D for the formation of rebaudioside A andrebaudioside M, respectively.

Example 74 Beta-1,3-ADP-Glycosyltransferase Variants of SEQ ID NO: 7388

In this Example, experiments for evolution and screening ofβ1,3-glycosyltransferase (β1,3GT) polypeptides derived from SEQ ID NO:7388 for improved glucosylation of steviol glycosides using in situsynthesized ADP-glucose are described. Directed evolution of the GTencoded by SEQ ID NO: 7387 (i.e., SEQ ID NO: 7388) was carried out byconstructing libraries of variant genes in which mutations associatedwith improved production identified during the development of thepresent invention were recombined and in which certain structuralfeatures were subjected to saturation mutagenesis. These libraries werethen plated, grown, and screened using the high-throughput (HTP) assaydescribed below to provide a twenty-second round (“Round 22”) of 88engineered GT variant polypeptides with glucosyltransferase activitytoward ADP-glucose and steviol glycosides.

HTP Assay for Glucose Transfer from Sucrose to ADP and then fromADP-Glucose to Rebaudioside A 60

Assays were performed on 96-well plates of cleared E. coli culturelysates expressing SEQ ID NO: 7387 variants. Pellets were lysed, andlysate was cleared as described in Example 60, and then diluted 20× into50 mM potassium phosphate buffer, pH 6. In order to thermally challengethe lysates, they were pre-incubated at 75° C. in a Thermotron®titre-plate shaker with 300 RPM shaking for 1 h. Assays were conductedwith 10 μL pre-incubated lysate in 100 μL reactions and with 20 g/Lrebaudioside A 60% substrate, 0.05 g/L ADP (Amresco, ultra pure grade)co-substrate, 0.05 g/L SUS SFP SEQ ID NO: 2510, 0.08 g/Lβ-1,2-glycosyltransferase (β12GT) SEQ ID NO: 7324, and 40 g/L sucrose(cane sugar). The following reaction conditions were used: 50 mMpotassium phosphate buffer, pH 6, 60° C. in a Thermotron® titre-plateshaker with 300 RPM shaking for 16 h. Reactions were solubilized by 20×dilution with water, quenched by 5× dilution with acetonitrile with 0.2%formic acid, and diluted 20× with water for analysis. Samples wereanalyzed by RapidFire-MS/MS as described in Example 31, Table 31.1.Glycosyltransferase variant polypeptides that produced rebaudioside Mfrom rebaudioside A 60 with in situ synthesized ADP-glucose at greaterquantities than SEQ ID NO: 7388 were identified. The engineeredpolypeptides are listed in Table 74.1 and Table 74.2. Shake-flask scalecultures were grown, lysed, cleared, and lyophilized to powder asdescribed in Example 31 for analysis of variants shown in Table 74.3relative to SEQ ID NO: 7388.

TABLE 74.1 β1, 3GT Round 22 Combinatorial Variants and RebM Levels SEQAmino Acid ID NO: Differences Increased (nt/aa) (Relative to SEQ ID NO:7388) RebM^(a) 7945/7946 M87K/M144Q + 7947/7948 K53E/W233Q/Q269R/ ++V3901/V3951/Y396T 7949/7950 V9G/M144Q/K331R + 7951/7952 M87K/M144Q/Y396T++ 7953/7954 K53E/M144Q/W233Q/ ++ Q269R/K331R/K4281 7955/7956 Q269R ++7957/7958 R37S/W233S + 7959/7960 M183L/W233Q/R234N/K331R/K428I +7961/7962 V9G/R37S/S113D/Y396T ++ 7963/7964 V9G/M87K + 7965/7966R37S/S113D/M144Q/Y396T + 7967/7968 Q269R/K4281/I437L ++ 7969/7970S113D/W233Q/R234N + 7971/7972 W233Q/Y396T/N399Q + 7973/7974 K53E/R234N++ 7975/7976 M144Q/R234N/Q269R + 7977/7978 D134E/K222A/V263T ++7979/7980 V263T ++ 7981/7982 D69A/D134E ++ 7983/7984D69A/V263T/D434E/A4385/P439H + 7985/7986 K171P/V263T ++ 7987/7988L3225 + 7989/7990 D69A/V263T ++ 7991/7992 H81T +++ 7993/7994K222A/V263T/R4351/5442F + 7995/7996 H81T/K222A/V263T/L322S/R435E/S442F++ 7997/7998 H81T/D134E +++ 7999/8000 H81T/L433A/R435I/A438R/S442F ++8001/8002 D69A +++ 8003/8004 P2K/N3M/L433A/R435E/5442F + 8005/8006D69A/H81T + 8007/8008 H81T/K222A +++ 8009/8010 K222A +++ 8011/8012P2K/H81T + 8013/8014 D69A/P439H +++ 8015/8016S61Q/L243I/D300A/R308L/V407I/R411T + 8017/8018C156S/Y165L/H248W/D300A/Q303V/R308L ++ 8019/8020L120V/C156S/Q159K/H248W/D300A/R308L +++ 8021/8022L120V/Q159K/Q197K/V365I/R411T + 8023/8024L120V/S253T/D300A/Q303V/R308L/V407I + 8025/8026S61Q/C156S/Q159K/L163A/Y165L/L2431/ ++ H248W/S253T/D300A/Q303V/R308L8027/8028 R308L + 8029/8030 A85V/C156S/Q159K/L2431/H248W/ +++S253T/R308L/E405P/V407I/R411T 8031/8032 S61Q/L120V/C156S/L163A/ +Y165L/Q197K/Q303V/R308L 8033/8034 S61Q/C156S/Q197K/S253E +++ 8035/8036Q197K/D300A/R308L/R411T ++ 8037/8038 Q159K/L163A/Y165L/Q197K/S214R/ +L2431/D300A/Q303V/R308L/V407I 8039/8040 C156S/S214R/R308L/R411T +++8041/8042 S61Q/S214R + 8043/8044 L120V/Q159K/Q197K/R308L/V407I/R411T ++8045/8046 S61Q/C156S/L163A/L2431/H248W/ +++ D300A/Q303V/R308L/V407I8047/8048 S61Q/C156S/L163A/D300A/V365I/R411T + 8049/8050L120V/C156S/Q159K/E169D/Q197K/S214R/ ++ Q303V/R308L/V365I/E405P/V407I8051/8052 L120V/R308L/V407I/R411T + 8053/8054 C156S/H248W/S253T/R308L+++ 8055/8056 L120V/C156S/H248W/Q303V/R308L/R411T ++ 8057/8058Q197K/S253T/R308L/V407I + 8059/8060 S61Q/L120V/D300A/Q303V/V407I +++8061/8062 L120V/Q159K/Y165L/Q197K ++ 8063/8064 S61Q/V365I/E405P +8065/8066 L120V/Q197K/S253T/D300A/R308L + 8067/8068S61Q/S214R/D300A/R308L + 8069/8070 L163A/Q197K/S253E/D300A/ +Q303V/R308L/V365I 8071/8072 L120V/Q159K + 8073/8074S61Q/L163A/D300A/Q303V/ +++ R308L/E405P/V407I/R411T 8075/8076S61Q/Y165L/H248W/S253T/V407I/R411T + 8077/8078 S61Q/D300A/Q303V/R308L+++ 8079/8080 C156S/Q197K/H248W/D300A/R411T ++ 8081/8082D300A/Q303V/R308L + 8083/8084 D300A/R308L/E405P/R411T + 8085/8086Q197K/D300A/Q303V/R308L/V365I + 8087/8088S61Q/L120V/Q159K/D300A/R308L/V407I +++ 8089/8090S61Q/D300A/Q303V/E405P + ^(a)Levels of increased production weredetermined relative to the reference polypeptide of SEQ ID NO: 7388, anddefined as follows: “+” = at least 1.19-fold, but less than 1.26-fold;“++” = at least 1.26-fold, but less than 1.37-fold; and “+++” = at least1.37-fold increased production, as compared to the referencepolypeptide.

TABLE 74.2 β1, 3GT Round 22 Saturation Mutagenesis Variants and RebMLevels Amino Acid SEQ Differences ID NO: (Relative to SEQ Increased(nt/aa) ID NO: 7388) RebM^(a) 8339/8340 R76S + 8341/8342 A107L ++8343/8344 C338V ++ 8345/8346 T72P +++ 8347/8348 S61D +++ 8349/8350L56D + 8351/8352 A107V ++ 8353/8354 S61E ++ 8355/8356 R88M + 8357/8358R88L ++ 8359/8360 C156S + 8361/8362 A41E + 8363/8364 K139N + 8365/8366V407T + 8367/8368 M87E + ^(a)Levels of increased production weredetermined relative to the reference polypeptide of SEQ ID NO: 7388, anddefined as follows: “+” = at least 1.04-fold, but less than 1.14-fold;“++” = at least 1.14-fold, but less than 1.18-fold; and “+++” = at least1.18-fold increased production, as compared to the referencepolypeptide.SFP Characterization Assay and Analysis for Glucosyl Transfer fromSucrose to ADP and then from ADP-Glucose to Stevioside and RebaudiosideD

Shake flask powders (SFP) were reconstituted to a concentration of 20g/L and diluted to 0.005-0.15 g/L SFP in 100 μL total reaction volumesof 50 mM potassium phosphate buffer, pH 6, 15 mM stevioside (>95%purity) or rebaudioside D, 0.1 g/L ADP (Amresco, ultra pure grade)co-substrate, 0.05 g/L SUS SFP SEQ ID NO: 2510, and 37.5 mM sucrose(cane sugar). The reactions were incubated in a Thermotron® titre-plateshaker with 300 RPM shaking for 4 hours at 60° C. The reactions weresolubilized by 20× dilution with water, quenched by 5× dilution withacetonitrile with 0.2% formic acid, cleared by centrifugation, anddiluted 15× with water for analysis. Glycosylated products were detectedin by SPE-QQQ as described in Example 31, Table 31.1. A furtherthermostability characterization was conducted with the clarified shakeflask lysates prior to lyophilization as follows: lysates were diluted20× in phosphate buffer and incubated in a thermocycler at a gradient of60-75° C. for 24 h. To determine the percent of activity remaining, thepre-incubated lysates were assayed with 20 g/L rebaudioside A 60, 0.05g/L ADP, 40 g/L sucrose, 0.05 g/L SUS SFP SEQ ID NO: 2510, and 0.08 g/Lβ1,2GT SFP SEQ ID NO: 7324, in 50 mM potassium phosphate buffer at pH 6,with 16 h incubation at 60° C. in a Thermotron® titre-plate shaker with300 RPM shaking. Reactions were solubilized, quenched, and diluted asdescribed for the high throughput assay. The percent activity remainingwas expressed as production at the high temperature divided byproduction at the lowest pre-incubated temperature.

TABLE 74.3 β1, 3GT Round 22 Shake Flask Powder Variants and RebA, RebMfrom RebD, and RebM from RebA60, and Thermostability Levels Amino AcidIncreased SEQ Differences Increased Increased RebM (from ID NO:(Relative to RebM, RebA, A60), % Activity (nt/aa) SEQ ID NO: 7388) 60°C.^(a) 60° C.^(a) 60° C.^(a) Remaining^(b) 7947/7948K53E/VV233Q/Q269R/ + + ++ − V390I/V395I/Y396T 7949/7950 V9G/M144Q/K331R− ++ + − 7951/7952 M87K/M144Q/Y396T + +++ ++ − 7967/7968Q269R/K428I/I437L + − + − 7977/7978 D134E/K222A/V263T + + ++ + 8007/8008H81T/K222A ++ ++ ++ + 8029/8030 A85V/C156S/Q159K/ +++ − +++ +L243I/H248W/S253T/ R308L/E405P/V407I/R411T 8033/8034 S61Q/C156S/ ++ ++++ ++ Q197K/S253E 8039/8040 C156S/S214R/ ++ ++ ++ − R308L/R411T8059/8060 S61Q/L120V/D300A/ + + + + Q303V/V407I 8087/8088S61Q/L120V/Q159K/ ++ +++ +++ + D300A/R308L/V407I ^(a)Levels of increasedproduction were determined relative to the reference polypeptide of SEQID NO: 7388, at 0.019 g/L shake flask powder and defined as follows: “−”= production less than that of the reference polypeptide; “+” =production at least that of the reference polypeptide, but less than1.21-fold; “++” = at least 1.21-fold, but less than 1.45-fold; and “+++”= at least 1.45-fold increased production, relative to the referencepolypeptide. ^(b)The percent of activity remaining for each variant wasdetermined following 24 h pre-incubation at 73.5° C., relative to theproduction from each variant following 24 h pre-incubation at 60° C. andis defined as follows: “−” = less than 33% of activity remainedfollowing 24 h pre-incubation at 60° C. “+” = at least 33%, but lessthan 50% of the activity remained; and “++” = at least 50% activityremained.

In these experiments, all eleven variants in Table 74.3 produced morerebaudioside M from rebaudioside A 60 than SEQ ID NO: 7388, and eightvariants also produced more rebaudioside A from stevioside andrebaudioside M from rebaudioside D. SEQ ID NOS: 8034 and 8088 performedthe best overall. SEQ ID NO: 8088 was selected as the best enzyme forthe catalysis of glycosyltransfer from ADP-glucose to stevioside andrebaudioside D for the formation of rebaudioside A and rebaudioside M,respectively, because of its superior performance in the one-pot assay.

Example 75 Beta-1,3-ADP-Glycosyltransferase Variants of SEQ ID NO: 8088

In this Example, experiments for evolution and screening ofβ1,3-glycosyltransferase (β1,3GT) polypeptides derived from SEQ ID NO:8088 for improved glucosylation of steviol glycosides using in situsynthesized ADP-glucose are described. Directed evolution of the GTencoded by SEQ ID NO: 8087 (i.e., SEQ ID NO: 8088) was carried out byconstructing libraries of variant genes in which mutations associatedwith improved production identified during the development of thepresent invention were recombined and in which certain structuralfeatures were subjected to saturation mutagenesis. These libraries werethen plated, grown, and screened using the high-throughput (HTP) assaydescribed below to provide a twenty-third round (“Round 23”) of 80engineered GT variant polypeptides with glucosyltransferase activitytoward ADP-glucose and steviol glycosides.

HTP Assay for Glucose Transfer from Sucrose to ADP and then fromADP-Glucose to Rebaudioside A 60

Assays were performed on 96-well plates of cleared E. coli culturelysates expressing SEQ ID NO: 8087 variants. Pellets were lysed, andlysate was cleared as described in Example 34, and then diluted 10× into50 mM potassium phosphate buffer, pH 6. In order to thermally challengethe lysates, they were pre-incubated at 75° C. in a Thermotron®titre-plate shaker with 300 RPM shaking for 1 h. Assays were conductedwith 10 μL pre-incubated lysate in 100 μL reactions and with 20 g/Lrebaudioside A 60% substrate, 0.025 g/L ADP (Amresco, ultra pure grade)co-substrate, 0.05 g/L SUS SFP SEQ ID NO: 8420, 0.08 g/Lβ-1,2-glycosyltransferase (β12GT) SEQ ID NO: 7784, and 40 g/L sucrose(cane sugar). The following reaction conditions were used: 50 mMpotassium phosphate buffer, pH 6, 60° C. in a Thermotron® titre-plateshaker with 300 RPM shaking for 16 h. Reactions were solubilized by 20×dilution with water, quenched by 5× dilution with acetonitrile with 0.2%formic acid, and diluted 20× with water for analysis. Samples wereanalyzed by RapidFire-MS/MS as described in Example 31, Table 31.1.Glycosyltransferase variant polypeptides that produced rebaudioside Mfrom rebaudioside A 60 with in situ synthesized ADP-glucose at greaterquantities than SEQ ID NO: 8088 were identified. The engineeredpolypeptides are listed in Table 75.1 and Table 75.2. Shake-flask scalecultures were grown, lysed, cleared, and lyophilized to powder asdescribed in Example 1 for analysis of variants shown in Table 75.3relative to SEQ ID NO: 8088.

TABLE 75.1 β1, 3GT Round 23 Combinatorial Variants and RebM Levels SEQID NO: Amino Acid Differences Increased (nt/aa) (Relative to SEQ ID NO:8088) RebM^(a) 8481/8482 M87K/Q91L/V120L/W233Q + 8483/8484M87K/Q91L/K94C/V263T/E389L + 8485/8486 M87A/Q91L/K94C/W233Q/E259T +8487/8488 M87K/Q91L/K94C + 8489/8490 Q91L/K94C/W233Q/E259T + 8491/8492M87A/Q91L/V120L/W233Q + 8493/8494 M87K/Q91L/W233Q + 8495/8496 Q91L/V120L++ 8497/8498 W233Q/E259T/E389L +++ 8499/8500 Q91L/W233Q ++ 8501/8502V263T/E389L + 8503/8504 Q91L/W233Q/E389L +++ 8505/8506 K94C/W233Q/R411T+++ 8507/8508 M87A/R435E ++ 8509/8510 Q91L/W233Q/E259T/E389L ++8511/8512 L163A/W233Q + 8513/8514 M87K/Q91L/K94C/V120L + 8515/8516M87A/K94C ++ 8517/8518 M87A +++ 8519/8520 M87A/Q91L/L163A ++ 8521/8522W233Q/A438S + 8523/8524 E259T + 8525/8526 M87A/Q91L/K94C/W233Q ++8527/8528 W233Q/E259T/V263T ++ 8529/8530 Q91L/V120L/W233Q + 8531/8532M87A/K4281/D431M/R435E ++ 8533/8534 V263T +++ 8535/8536 L322S +8537/8538 M87A/Q91L/V120L/W233Q/R411T/D431M/R435E/I437L ++ 8539/8540M87K/Q91L/L163A/W233Q/V263T +++ 8541/8542Q91L/K94C/V120L/W233Q/E389L/D431M/A438S + 8543/8544M87A/Q91L/E259T/V263T/E389L/K4281/D431M/R4351/I437L +++ 8545/8546M144Q/E389L + 8547/8548 M87A/W233Q/L322S/E389L/R411T + 8549/8550M87K/K94C/M144Q/V263T/K428I/R435E +++ 8551/8552 Q91L/K94C ++ 8553/8554M87K/Q91L + 8555/8556 E389L ++ 8557/8558 M87K +++ 8559/8560Q91L/K94C/V120L/W233Q ++ 8561/8562 W233Q +++ 8563/8564 K4281/D431M/R435E++ 8565/8566 M87A/Q91L/L322S + 8567/8568 M87K/Q91L/V263T/E389L ++8569/8570 Q91L ++ 8571/8572 M87A/W233Q/E389L +++ 8573/8574M87K/L163A/W233Q ++ 8575/8576 M87K/Q91L/L163A/E389L ++ 8577/8578M87A/W233Q +++ 8579/8580 M87K/Q91L/K94C/W233Q + 8581/8582M87K/Q91L/E389L +++ 8583/8584 M87A/Q91L ++ 8585/8586 M87A/Q91L/W233Q +++8587/8588 M87K/Q91L/L163A/W233Q ++ 8589/8590 M87A/E259T + 8591/8592M87A/K94C/V263T + 8593/8594 M87K/Q91L/M144Q/E259T + 8595/8596M87A/Q91L/W233Q/E389L + 8597/8598 M87A/V263T + 8599/8600M87A/VV233Q/E259T + 8601/8602 T72P/R76S/A107L/C156S + 8603/8604K53E/H81T/H195Q/Q197K + 8605/8606 D69A/T72P/R76S/A107L + 8607/8608R37S/T72P/H195Q/K331R + 8609/8610 H195Q/Q197K + 8611/8612T72P/R76S/A107L/H195Q/Q197K + 8613/8614 R37S/T72P/R76S/H81T + 8615/8616T72P/Q269R + 8617/8618 Q197K + 8619/8620R37S/T72P/R76S/A107L/C156S/K331R + 8621/8622 H81T/A107L/H195Q +^(a)Levels of increased production were determined relative to thereference polypeptide of SEQ ID NO: 8088, and defined as follows: “+” =at least 1.3-fold, but less than 1.55-fold; “++” = at least 1.55-fold,but less than 1.8-fold; and “+++” = at least 1.8-fold increasedproduction, as compared to the reference polypeptide.

TABLE 75.2 β1, 3GT Round 23 Saturation Mutagenesis Variants and RebMLevels Amino Acid SEQ Differences ID NO: (Relative to SEQ Increased(nt/aa) ID NO: 8088) RebM^(a) 9223/9224 P324D + 9225/9226 V413L +9227/9228 V451Q ++ 9229/9230 P324G + 9231/9232 S252P + 9233/9234 S111T +9235/9236 S255T + 9237/9238 L328T + 9239/9240 S55G + ^(a)Levels ofincreased production were determined relative to the referencepolypeptide of SEQ ID NO: 8088, and defined as follows: “+” = at least1.05-fold, but less than 1.2-fold; and “++” = at least 1.2-foldincreased production, as compared to the reference polypeptide.SFP Characterization Assay and Analysis for Glucosyl Transfer fromSucrose to ADP and then from ADP-Glucose to Stevioside and RebaudiosideD

Shake flask powders (SFP) were constituted to a concentration of 20 g/Land diluted to 0.005-0.15 g/L SFP in 100 μL total reaction volumes of 50mM potassium phosphate buffer, pH6, 15 mM stevioside (>95% purity) orrebaudioside D, 0.1 g/L ADP (Amresco, ultra pure grade) co-substrate,0.05 g/L SUS SFP SEQ ID NO: 8420, and 37.5 m sucrose (cane sugar). Thereactions were incubated in a Thermotron® titre-plate shaker with 300RPM shaking for 4 hours at 60° C. The reactions were solubilized by 20×dilution with water, quenched by 5× dilution with acetonitrile with 0.2%formic acid, cleared by centrifugation, and diluted 15× with water foranalysis. Glycosylated products were detected in by SPE-QQQ as describedin Example 31, Table 31.1. A one-pot reaction was conducted with0.01-0.3 g/L SFP in 100 μL total reaction volumes of 50 mM potassiumphosphate buffer, pH6, 20 μL RebA60, 0.025 g/L ADP (Amresco, ultra puregrade) co-substrate, 0.05 g/L SUS SFP SEQ ID NO:8420, 0.12 g/L β1,2GTSFP SEQ ID NO:7784, and 40 g/L sucrose (cane sugar). The reactions wereincubated in a Thermotron® titre-plate shaker with 300 RPM shaking for16 hours at 60° C. The reactions were solubilized by 20× dilution withwater, quenched by 5× dilution with acetonitrile with 0.2% formic acid,cleared by centrifugation, and diluted 20× with water for analysis.

TABLE 75.3 β1, 3GT Round 23 Shake Flask Powder Variants and RebA, RebMfrom RebD, and RebM from RebA60, and Thermostability Levels IncreasedSEQ Increased Increased RebM ID NO: Amino Acid Differences RebM, RebA,(from A60), (nt/aa) (Relative to SEQ ID NO: 8088) 60° C.^(a) 60° C.^(a)60° C.^(a) 8491/8492 M87A/Q91L/V120L/W233Q ++ + ++ 8503/8504Q91L/W233Q/E389L + + ++ 8571/8572 M87A/W233Q/E389L + ++ + 8597/8598M87A/V263T ++ + ++ 8611/8612 T72P/R76S/A107L/H195Q/Q197K ++ − +++8621/8622 H81T/A107L/H195Q + − ++ ^(a)Levels of increased productionwere determined relative to the reference polypeptide of SEQ ID NO:8088, at 0.019 g/L shake flask powder and defined as follows: “−” =production less than that of the reference polypeptide; “+” = productionat least that of the reference polypeptide, but less than 1.1-fold; “++”= at least 1.1-fold, but less than 1.25-fold; and “+++” = at least1.25-fold increased production, relative to reference polypeptide.

In these experiments, all eleven variants in Table 75.3 produced morerebaudioside M from rebaudioside A 60 than SEQ ID NO: 8088, and eightvariants also produced more rebaudioside A from stevioside andrebaudioside M from rebaudioside D. SEQ ID NO: 8598 was selected as thebest enzyme for the catalysis of glycosyltransfer from ADP-glucose tostevioside and rebaudioside D for the formation of rebaudioside A andrebaudioside M, respectively, because of its superior performance in theone-pot assay.

Example 76 Thermal Tolerance of Engineered Glycosyltransferases andRecycling Enzyme

Three assays were performed to determine the thermal tolerance of anengineered β-1,3-glycosyltransferase (β1,3GT, SEQ ID NO: 6864),β-1,2-glycosyltransferase (β1,2GT, SEQ ID NO: 4550), and sucrosesynthase recycling enzyme (SUS, SEQ ID NO: 2510) using shake flaskpowders (SFP).

First, a multi-day stability assay at 60° C. was performed by dilutingeach enzyme SFP in 50 mM potassium phosphate pH 6 (β1,3GT, 0.5 g/L;β1,2GT, 0.25 g/L; SUS, 0.1 g/L) and incubating for variable times from0-48 h prior to assaying. The amount of activity remaining wasdetermined by assaying under the conditions described in this Example.For β1,3GT, 10 μL of the 0-48 h pre-incubated shake flask powder wasused in 100 μL total reaction volumes of 50 mM potassium phosphatebuffer, pH 6, 15 mM stevioside (>95% purity) or rebaudioside D, 0.2 mMADP (Amresco, ultra pure grade) co-substrate, 0.05 g/L SUS SFP SEQ IDNO: 2432, and 37.5 mM sucrose (cane sugar). The reactions were incubatedin a Thermotron® titre-plate shaker with 300 RPM shaking for 4 hours at60° C., then solubilized by 20× dilution with water, quenched by 5×dilution into acetonitrile with 0.2% formic acid, cleared bycentrifugation, and diluted 15× with water for analysis by SPE-QQQ asdescribed in Example 31, Table 31.1. At 28 h, 60% of the un-preincubatedactivity for rebaudioside D to rebaudioside M remained, and at 48 h, 39%activity remained; for stevioside to rebaudioside A, 34% remained after28 h and 19% remained after 48. Thus, the β1,3GT SEQ ID NO: 6864 has ahalf-life of 20-40 h at 60° C. For β1,2GT, the assay was performedsimilarly with 20 mM rebaudioside A (>97% purity) and 50 mM sucrose. At24 h, 50-74% of the un-preincubated activity remained, and at 46 h, 33%of the activity remained. Thus, the β1,2GT SEQ ID NO: 4550 has ahalf-life of 29-58 h at 60° C. For SUS, the assay was performedsimilarly with 15 mM rebaudioside A (>97% purity), 37.5 mM sucrose, 9 mMfructose, and 0.5 g/L β1,2GT SEQ ID NO: 4550 as the coupled enzyme. At24 h, 89% of the un-pre-incubated activity remained, and at 48 h, 86% ofthe activity remained. Thus, the SUS SEQ ID NO: 2510 has a half-lifeof >100 h at 60° C. All three of these half-lives at 60° C. represent alarge difference from wild-type enzymes, which do not have significantstability above ambient temperature.

Second, identical stocks of each of the three enzymes were preincubatedfor 24 h across a temperature gradient of 60-69.1° C. (SUS), 59.9-79.9°C. (β1,2GT), or 59.7-75.1° C. in a thermocycler, and enzymes wereassayed as described above to determine the activity remaining relativeto the lowest pre-incubation temperature (˜60° C. for all three). Forβ1,3GT, 20% of the activity was retained following pre-incubation at73.5° C. relative to preincubation at 59.7° C. For β1,2GT, 17% of theactivity was retained following pre-incubation at 73.6° C. For SUS, 85%of the activity was retained following pre-incubation at 69.1° C.Stability at 24 h up to temperatures >70° C. allows for a wide range offermentation downstream processing temperatures and for a wide range ofsteviol glycoside conversion temperatures. By heating the steviolglycoside conversion reaction, the risk of microbial contamination isreduced and steviol glycoside substrate and product solubilities areenhanced, increasing the rate of conversion. Additionally, the intrinsicreaction rate is slightly increased by increasing temperature.

Third, β1,3GT, 0.1 g/L; β1,2GT, 0.025 g/L; and SUS, 0.01 g/L wereassayed in single substrate conversion assays as described above withoutpre-incubation with incubation at 55-65° C. to determine whether theenzymes would be robust in this temperature range. For all threeenzymes, there was <36% increase or decrease in the conversion measuredafter 4 h at 65° C. relative to 55° C.

Example 77 Assay of Engineered Glycosyltransferases and Recycling Enzymewith Additional NDPs and NDP-Glucoses

Multiple nucleoside diphosphates (NDPs) and nucleosidediphosphate-glucoses (NDP-glucoses) can be used with the engineeredglycosyltransferases and recycling system.

To determine the promiscuity of the engineered β-1,3-glycosyltransferase(β1,3GT, SEQ ID NO: 6864) and β-1,2-glycosyltransferase (β1,2GT, SEQ IDNO: 4550) for alternate NDP-glucose donors, three commercially availableNDP-glucoses were tested: ADP-glucose (Amresco, ultra high grade),GDP-glucose (Sigma, >97% purity), and TDP-glucose (Carbosynth, >95%purity) with enzyme shake flask powders (SFP). For β1,3GT, the reactionswere carried out with 0.025 g/L SFP in 1 mM stevioside (>95% purity), 1mM NDP-glucose, 50 mM potassium phosphate buffer, pH 6. At 1, 2, and 3h, the reactions were solubilized by 4× dilution in water, quenched by5× dilution in acetonitrile with 0.2% formic acid, and diluted 5× inwater for analysis. For β1,2GT, the reactions were carried out with0.0025 g/L SFP in 1 mM rebaudioside A (>97% purity), but were otherwiseidentical to the method described above. The results are summarized inTable 68.1 as the percent (%) conversion of steviol glycoside substratein the first hour.

TABLE 77.1 NDP-Glucose Utilization by Engineered GlycosyltransferasesSEQ ID NO: ADP- GDP- TDP- (nt/aa) Glucose^(a) Glucose^(a) Glucose^(a)4550 ++++ +++ ++ 6864 + + − ^(a)Levels of NDP-glucose utilization weredetermined as mmol steviol glycoside converted per gram shake flaskpowder and defined as follows: “−” = activity less than 10 mmol/g; “+” =activity between 10 and 30 mmol/g; “++” = activity between 30 and 50mmol/g; “+++” = activity between 50 and 70 mmol/g; and “++++” = at least70 mmol/g activity.

To determine the promiscuity of the engineered β-1,2-glycosyltransferase(β1,2GT, SEQ ID NO:4550) and sucrose synthase recycling enzyme (SUS, SEQID NO:2510) for alternate NDP co-factors, four commercially availableNDPs were tested: ADP (Sigma, >95%), CDP (Sigma, >95%), GDP(Sigma, >96%), IDP (Sigma, >96%). The reactions were carried out with0.001 g/L SUS SFP and 0.1 g/L β1,2GT SFP in 10 mM rebaudioside A (>97%purity), 0.2 mM NDP, 50 mM potassium phosphate buffer, pH6. At 1,2, and3h, the reactions were solubilized by 40× dilution in water, quenched by5× dilution in acetonitrile with 0.2% formic acid, and diluted 5× inwater for analysis. A second sucrose synthase (SEQ ID NO: 72) was alsotested, using 10 μL of purified protein glycerol stock in the 100reaction. The results are summarized in Table 77.2 as NDP turnovers(mmol rebaudioside D/mmol cofactor) in the first hour. As these dataindicate the use of ADP/ADP-glucose provides useful reaction conditions.In addition, these compounds are beneficial from an economicperspective, as compared to other options (e.g., UDP/UDP-glucose).

TABLE 77.2 NDP Utilization by Engineered Glycosyltransferase andRecycling Enzyme SEQ ID NO: (nt/aa) ADP^(a) CDP^(a) GDP^(a) IDP^(a) 2510+++ − − −  72 ++ − + + ^(a)Levels of NDP utilization were determined asmmol steviol glycoside converted per mmol cofactor in 1 h and defined asfollows: “−” = activity less than 1; “+” = between 1 and 2; “++” =between 2 and 3; and “+++” = greater than 3 cofactor turnovers.

Example 78 Process to Convert Rebaudioside A 60% to Rebaudioside M

Reb A 60 (˜1:2 mixture of stevioside and rebaudioside A [reb A] by mass)has unexpectedly high solubility in water and sucrose solutions. Theindividual aqueous solubility of stevioside and Reb A is reported to bein the range of 3-5 g/L. Surprisingly, 100-200 g/L solutions of Reb A 60were prepared either in water or in 200 g/L of sucrose and noprecipitate developed in said solutions upon standing at roomtemperature for one week. Remarkably, during the course of thereactions, both the Reb D intermediate and the Reb M product remainedsoluble (as evident by the homogenous reaction mixture) at levels (ca.30-50 g/L) far exceeding their reported solubility limits (˜0.3-0.5 and3-5 g/L, respectively). The efficiency of the process was greatlyenhanced by the unpredictably high solubility of the Reb A 60 startingmaterial and the Reb D intermediate.

A process to convert Reb A 60 to rebaudioside M was developed with anengineered β-1,3-glycosyltransferase (β1,3GT, SEQ ID NO: 6138),β-1,2-glycosyltransferase (β1,2GT, SEQ ID NO: 3696), and sucrosesynthase (SUS, SEQ ID NO: 1846). A recycling stock solution wasprepared, consisting of 0.2 g/L SUS, 0.1 g/L ADP, and 200 g/L sucrose in50 mM pH 6, potassium phosphate buffer. β1,2GT was dissolved in thissolution to 1.6 g/L, and a separate stock of β1,3GT was dissolved in therecycling stock solution to 2.0 g/L. Then, 100 mg of Reb A 60 was placedin a one dram vial and 0.5 mL each of the 1.6 g/L β1,2GT stock and 2.0g/L β1,3GT stock were added. The resulting homogenous solution wasstirred at 55° C. Precipitate gradually developed and at 24 h thereaction mixture was a thick white slurry. HPLC analysis showed thepresence of 90-94% Reb M (˜120-130 g/L Reb M).

In another experiment, the above reaction was carried out in thepresence of 0.5 g/L of ADP and 10 mM of EDTA. Under these conditions,the β1,2GT and β1,3GT loadings were both decreased to 0.4 g/L, whilestill reaching >90% conversion to Reb M. When the lyophilized enzymepowders were produced at 10-15 L scale fermentation and processeddownstream with 60° C. heat treatment and ultrafiltration, 95% of thestevioside and rebaudioside A in Reb A 60 were converted to rebaudiosideM under certain ratios of β1,2GT stock to β1,3GT stock.

A process to convert Reb A 60 to rebaudioside M was developed withanother set of engineered β-1,3-glycosyltransferase (β1,3GT, SEQ ID NO:6864), β-1,2-glycosyltransferase (β1,2GT, SEQ ID NO: 4550), and sucrosesynthase (SUS, SEQ ID NO: 2510) enzymes. A recycling stock solution wasprepared, consisting of 0.2 g/L of SUS, 0.5 g/L of ADP and 200 g/L ofsucrose in 50 mM pH 6, potassium phosphate buffer with 10 mM of EDTA.β1,2GT was dissolved in this solution to 0.6 g/L, and a separate stockof β1,3GT was dissolved in the recycling stock solution to 1.2 g/L.Then, 100 mg of Reb A 60 was placed in a one dram vial and 0.5 mL eachof the 0.6 g/L β1,2GT stock and 1.2 g/L β1,3GT stock were added. Theresulting homogenous solution was stirred at 60° C. Precipitategradually developed and at 24 h the reaction mixture was a thick whiteslurry. HPLC analysis showed the presence of 90-92% Reb M (˜120-130 g/LReb M). This reaction was scaled to 2 grams Reb A 60 in 20 mL with 0.6g/L β1,2GT, 0.2 g/L SUS, and 1.2 g/L β1,3GT with 0.5 g/L ADP and 10 mMEDTA at 60° C., and it was determined that 87.1% RebM was obtained byarea under curve using JECFA method. For purification, the reaction mixwas centrifuged at 40° C., the supernatant was decanted, and the pelletwas resuspended with 1 volume deionized water, centrifuged at 20° C.,and the supernatant was decanted. This wash was repeated for four totalwashes and the pellet was lyophilized, yielding 94.3% RebM as determinedby area under curve using JECFA method.

All publications, patents, patent applications and other documents citedin this application are hereby incorporated by reference in theirentireties for all purposes to the same extent as if each individualpublication, patent, patent application or other document wereindividually indicated to be incorporated by reference for all purposes.

While various specific embodiments have been illustrated and described,it will be appreciated that various changes can be made withoutdeparting from the spirit and scope of the invention.

We claim:
 1. An engineered sucrose synthase comprising a polypeptidesequence that is at least 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%,89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequenceidentity to SEQ ID NO:72, 74, 1080, 1158, 1222, 1392, 1456, 1582, 1764,1804, 1840, 2064, 2432, 2510, 7506, and/or
 8420. 2. The engineeredsucrose synthase of claim 1, wherein said polypeptide sequence of saidengineered sucrose synthase comprises at least one mutation or mutationset at one or more positions selected from 4/9/349/532,4/13/113/343/532, 4/13/113/532, 4/33/47/52/343/532, 4/47/52/532,4/113/532, 4/13/113, 4/13/532, 4/33/113, 4/343, 7, 8, 44, 95, 117/440,136, 221, 343/532, 440, 444, 478, 532, 583, 611, 615, 615/789, 695, 722,and 788, wherein said positions are numbered with reference to SEQ IDNO:74.
 3. The engineered sucrose synthase of claim 1, wherein saidpolypeptide sequence of said engineered sucrose synthase comprises atleast one mutation or mutation set at one or more positions selectedfrom 8/221, 47/221, 68/129/248, 68/129/248/595/600/756,68/146/248/387/506/550, 68/189/272/316/477/719/756,75/105/154/215/264/345, 75/105/345/410/769, 75/105/530, 75/345/530,85/170/225/266/534, 87/125/230/267/375/464/708,93/129/506/550/595/719/756, 93/477/635, 95/136/788,95/201/478/583/724/788, 95/385/478/583/788, 95/440/478/724/788/792,95/444/478/603/792, 95/444/478/724/788, 95/478/724, 98/250,113/225/266/415, 126/314/499/549/589/755, 136/440/444/478/603,136/440/444/478/583/788, 136/444/478/583/788/792, 225/372/534, 266,306/358/703/776, 358/636/737, 440/444/583/724/788, 440/478, and 466,wherein said positions are numbered with reference to SEQ ID NO:1080. 4.The engineered sucrose synthase of claim 1, wherein said polypeptidesequence of said engineered sucrose synthase comprises at least onemutation or mutation set at one or more positions selected from8/68/95/98/358/478/595/724/792, 8/68/98/221/248/250/440/477/534/595/724,8/68/788, 8/93/95/98/136/221/595/600/788,8/93/95/113/250/440/595/600/724/788, 8/95/98/440/478/534/600/788,8/136/248/478/788, 47/75/85/105/125/129/170/635,47/75/85/105/375/756/776, 47/75/85/264/267/372/415/635,47/75/85/87/129/375/776, 47/75/85/87/170/372/756,47/85/105/129/201/230/267/583, 47/85/125/372/583/635/756, 47/85/170/756,47/85/87/105/125/635, 47/85/87/154/756, 47/125/129/375/756/776,47/129/170/635, 47/154/372/375/583/635/708/756,68/93/95/358/440/444/478/534/595/603, 68/93/95/444/788,68/93/98/136/248/250/358/440/534/724, 75/85/87/105/264/267/583/708,75/85/129/154/264/375, 85/125/215/375/415/635/776,85/87/105/215/267/756, 85/87/129/375/756/776, 87/125/129/170/230/756,87/154/306/375/756, 93/95/98/534/792, 95/440/444/724/788,129/215/372/756, and 170/264/267, wherein said positions are numberedwith reference to SEQ ID NO:1158.
 5. The engineered sucrose synthase ofclaim 1, wherein said polypeptide sequence of said engineered sucrosesynthase comprises at least one mutation or mutation set at one or morepositions selected from 47/68/93/98/358/440, 47/68/154/372/375,47/93/98/136/154/772/776, 47/93/98/154/372/375/776,47/93/98/358/583/635, 47/93/129/136/154/250/372/534/635/724,47/93/129/136/375/534/583, 47/93/358/372/375/440/724,47/93/358/372/375/776, 47/98/129/358/372/375/438/534,47/98/129/375/534/635/724/776, 47/98/372/375, 47/125/154,47/129/136/372/375/534, 47/129/248/250/372/375/534/724, 47/136/583/776,47/358/440/724, 47/358/635/776, 47/372/635/776,68/93/98/129/358/375/724, 68/93/154/358/372/440/776, 68/129/440,68/129/583/724, 68/136/724, 68/154/358/375, 68/154/534/635,68/375/440/534/724/776, 93/98/125/154/248, 93/98/125/154/250/440,93/98/129/154/248, 93/98/154/250/358/375/534, 93/98/154/635/776,93/98/534, 93/125/154/440/534, 93/129/250/358/372/375/583,93/154/248/724, 93/358/534/635, 98/129/375/583, 98/534/583/635,129/136/154/248/250/372/375/534, 129/136/375,129/154/248/250/358/375/534/635, 129/358/372/635, 154/250/358/375/583,154/358/375/534/776, 154/534/635/724, 372/375/776, 375/635, and 534/583,wherein said positions are numbered with reference to SEQ ID NO: 1222.6. The engineered sucrose synthase of claim 1, wherein said polypeptidesequence of said engineered sucrose synthase comprises at least onemutation or mutation set at one or more positions selected from17/357/364/434/519/684, 17/357/434/519/684, 17/434/684, 17/684,54/97/118/307/694/727/738, 68/98/129/136, 68/98/129/136/154,68/98/129/136/154/534, 68/98/129/154/534, 68/98/129/154/635,68/98/136/154/534/635, 68/98/136/154/635, 68/98/154, 68/98/154/534,68/98/154/534/635, 68/129/136, 68/129/136/154, 68/129/136/154/464/635,68/129/136/534/635, 68/129/154, 68/129/154/765, 68/136/154/534/635,68/136/534/635, 68/136/635, 68/154, 68/154/534/635, 68/154/635,97/118/442/694/727/738, 98/129/136/154, 98/129/136/154/635,98/129/136/534, 98/129/136/635, 98/129/154, 98/129/154/534/635,98/129/534/635, 98/136/154/635, 98/136/534/635, 98/136/635, 98/154,98/154/534, 122, 129/136, 129/136/154/635, 129/136/534, 129/136/635,129/154/534, 129/154/635, 129/635, 132/136/154/534/635, 136/154/635,136/534/635, 136/603, 136/635, 154, 154/635, 157, 160, 161, 167, 253,285, 381, 519, 550, 563, 564, 635, and 785, wherein said positions arenumbered with reference to SEQ ID NO:
 1392. 7. The engineered sucrosesynthase of claim 1, wherein said polypeptide sequence of saidengineered sucrose synthase comprises at least one mutation or mutationset at one or more positions selected from 17/54/97/136/329/550/684/738,17/54/97/329/524/684, 17/54/161/519/727/738, 17/54/524/550/727,17/161/434/524/766, 17/434/524/684, 17/434/738, 17/442/524/550/684/721,17/727, 17/738, 54/97/161/434/442, 54/97/434/524/550/684/727,54/136/442/550, 54/434/524/738, 97/136/519/550/727/738, and329/550/684/727/738, wherein said positions are numbered with referenceto SEQ ID NO:
 1456. 8. The engineered sucrose synthase of claim 1,wherein said polypeptide sequence of said engineered sucrose synthasecomprises at least one mutation or mutation set at one or more positionsselected from 14/137/356/745, 14/570, 14/570/745, 26/117/365,26/164/165/213/586, 71/158/222/356, 71/222/236, 71/319/356/606,117/158/213/332/608, 117/164/707, 117/213/365/517, 117/311/332, 117/608,122, 122/160/161/167/550, 122/160/161/282/381/550,122/160/161/282/381/550/636, 122/160/161/282/550, 122/160/161/550/636,122/160/167/282/381/550/636, 122/160/282/381, 122/160/282/381/550,122/160/282/550, 122/160/381/550, 122/160/381/550/636, 122/160/550,122/160/550/636, 122/161/550, 122/167, 122/167/550, 122/282/381/550,122/282/550, 122/282/550/636, 122/381/706, 122/550, 137/319/570,157/253/519, 160/161, 160/161/282/381/550, 160/161/282/550,160/161/550/636/735, 160/167/282/381/636, 160/282, 160/282/381/550,160/282/550/636, 160/381/550/636/681, 161/282/550/636, 161/381/550,165/311, 167/282/636, 167/550, 213/365/517/707, 236, 253/519,253/519/563, 253/519/635, 253/563/635, 270/322/517,270/367/452/517/613/700/750, 270/452/517/700/750, 270/570, 282/381/550,282/550, 356/570, 381/550, 517, 517/562/750, 517/640, 519/563, 550,550/636, and 562, wherein said positions are numbered with reference toSEQ ID NO:
 1582. 9. The engineered sucrose synthase of claim 1, whereinsaid polypeptide sequence of said engineered sucrose synthase comprisesat least one mutation or mutation set at one or more positions selectedfrom 63/536, 117/122/270/540/681, 181/536/548, 181/536/548/705,181/548/705, 270/681, 347/532, 347/536/548/705, 407/570/681, 407/681,536, 536/548, 536/548/699, 536/705, 548, 548/580, 548/705, 580, 681,699, and 705, wherein said positions are numbered with reference to SEQID NO:
 1764. 10. The engineered sucrose synthase of claim 1, whereinsaid polypeptide sequence of said engineered sucrose synthase comprisesat least one mutation or mutation set at one or more positions selectedfrom 13, 17, 18, 30, 37, 52, 57, 60, 71, 85, 87, 90, 98, 99, 118, 129,164, 180, 183, 347/434/517/562/640/681, 347/434/532/562/640/681,347/434/550/562/681, 347/434/681, 347/517/532/681,347/532/550/640/681/699, 347/536/562/681, 347/550/580/681, 347/550/681,347/681, 365, 388, 389, 415, 433, 434/517/532/681, 517/681, 531,532/681, 535, 536/580/681, 539, 562/681, 589, 606, 608, 707, 711, 727,738, 748, 765, 769, and 789, wherein said positions are numbered withreference to SEQ ID NO:
 1804. 11. The engineered sucrose synthase ofclaim 1, wherein said polypeptide sequence of said engineered sucrosesynthase comprises at least one mutation or mutation set at one or morepositions selected from 14, 15, 17/52/87/118/129/388/589/738/765,17/52/87/118/129/589/738, 17/52/87/118/129/589/738/765,17/52/87/118/129/589/765, 17/52/87/129/388/589,17/52/87/129/388/589/738, 17/52/87/129/738, 17/52/87/388/589/765,17/52/87/589/738/765, 17/52/118/129/265/589/765,17/52/118/129/388/589/738/765, 17/52/118/129/589/738/765,17/52/118/129/738/765, 17/52/118/388/589/738, 17/52/118/388/589/738/765,17/52/118/388/738/765, 17/52/129/388/589/738, 17/52/129/388/589/738/765,17/52/129/589, 17/52/129/589/738, 17/52/129/589/765,17/52/129/653/738/765, 17/52/129/738, 17/52/129/738/765,17/52/388/589/738, 17/52/388/589/738/765, 17/52/589/738/765,17/52/589/765, 17/87/118/388/738, 17/87/129/388/738,17/118/129/388/738/765, 17/129/589, 17/129/589/738, 17/129/589/738/765,17/129/738/765, 17/388/589/738, 17/589/738, 17/589/765, 17/738/765,18/362, 20, 24, 26, 33, 33/154, 46, 50, 52/84/129/388/738/765,52/87/118/129/388/765, 52/87/118/388/589/738, 52/87/118/589/738/765,52/87/129/388/738/765, 52/87/129/765, 52/87/589/738, 52/87/738,52/118/129/589/738, 52/118/129/765, 52/118/388/589/738,52/118/388/738/765, 52/129/589/738/765, 52/129/589/765, 52/129/738,52/388/738/765, 52/589/738, 52/589/738/765, 52/738/765, 54, 58, 59,59/72, 79, 81, 84/129/589/738/765, 87/118/129/765, 87/129/388/589, 92,93, 97/154, 104, 105, 130, 134, 154, 165, 175, 185, 212, 213, 218, 241,256, 263, 316, 319, 349, 360, 362, 364, 390, 393, 434, 480, 498, 530,534, 534/739, 542, 589/738/765, 603, and 652, wherein said positions arenumbered with reference to SEQ ID NO:
 1840. 12. The engineered sucrosesynthase of claim 1, wherein said polypeptide sequence of saidengineered sucrose synthase comprises at least one mutation or mutationset at one or more positions selected from 21, 25/112, 41,57/71/87/347/434/562/606, 57/71/129/180/434/536/562,57/71/129/434/531/536/562, 57/71/129/531/532/536/539/606,57/71/562/606/711/789, 57/71/789, 57/87/180/531/532/562/606/612/711,57/87/347/562, 57/90/129/562, 57/90/129/562/711,57/96/129/180/531/532/550/562, 57/129/347/531/532/539/562/711/747,57/129/347/536/550/562/711/789, 57/129/347/550/711,57/129/531/539/562/789, 57/129/536/606/789, 57/129/606, 57/180/562,57/180/562/606/612, 57/347/434/531/532/539/789,57/434/550/562/606/612/789, 57/531/532/536/562, 57/562/606/711,57/562/711, 71/129/180/347/531/539/550,71/129/180/434/532/536/539/711/789, 71/129/531, 71/129/606,71/347/532/550/562/711, 71/347/536/562/612/789, 71/536/539/562,87/189/532/536/562/711/789, 87/347/531/606/789, 87/347/536/539/550, 89,90/129/539/550/606, 91, 112, 129/180/434/562/711/789,129/180/606/711/789, 129/347/562, 129/536/539/562, 129/539/562/789,129/550, 129/550/562, 129/562/606/711, 180/532, 180/550/606, 186, 200,226, 259, 318, 330, 347/531/550/711, 347/536/539/550/711/789,347/536/562/606/612, 347/550/562/606, 434/531/539/550/562/711,434/550,485, 487, 531/532/536/539/562/711/789, 531/532/536/550/562/606/789,531/532/562/606/711, 532/539/550, 562/711, 641, 674, 684, 688, 763, and764, wherein said positions are numbered with reference to SEQ ID NO:2064.
 13. The engineered sucrose synthase of claim 1, wherein saidpolypeptide sequence of said engineered sucrose synthase comprises atleast one mutation or mutation set at one or more positions selectedfrom 25, 33/47/59/81/175/530/534/550/606,33/58/59/81/130/480/530/534/550/652, 33/58/59/480/530/534/550,33/58/154/480/534/550/603/606, 33/59/480/530/534/550/606,33/79/81/175/530/534, 33/79/81/175/530/534/603,33/79/154/480/530/534/550, 33/81/130/480/530/534/550,33/81/175/530/534/542/550/652, 33/130/530/534/550,33/154/480/530/534/603/606, 33/154/534, 33/530/534/550, 42,58/59/79/175/480/534/550/652, 59/154/530/534/550, 70, 75, 77,79/81/480/530/534/550/603/606/652, 81/480/530/534/550, 106,130/480/530/534/550/603/606, 199, 265, 267, 380, 410, 561, 642, and 758,wherein said positions are numbered with reference to SEQ ID NO: 2432.14. The engineered sucrose synthase of claim 1, wherein said polypeptidesequence of said engineered sucrose synthase comprises at least onemutation or mutation set at one or more positions selected from 7/12,12, 27, 29, 41, 41/71, 41/71/112, 41/71/112/259/485/487/684/688,41/71/112/259/485/688, 41/71/259/485/532, 41/71/485, 41/71/485/532/684,41/71/487, 41/71/487/532/684, 41/71/532, 41/71/532/684, 41/71/684,41/84/259/485/487, 41/91/112/485, 41/91/112/485/487/532/684,41/91/112/485/532/684, 41/91/485, 41/112, 41/112/259/485/487,41/112/259/487/532/684, 41/112/485/684, 41/112/487/684, 41/112/532,41/112/684/688, 41/259/485, 41/259/485/487, 41/259/485/487/532/684,41/259/485/487/684/688, 41/259/532, 41/485, 41/485/487,41/485/487/684/688, 41/485/532, 41/485/532/688, 41/485/684/688,41/487/684, 41/532, 41/684, 41/684/688, 44, 44/112/684/688, 45, 47, 48,51, 55, 71/112/259/485/487/684, 71/112/485/688, 71/485/684/688, 71/532,71/684/688, 72, 95, 100, 112, 112/259, 112/259/532/684/688,112/259/684/688, 112/485/684, 112/485/684/688, 116, 136, 139, 176, 178,198, 201, 205, 205/485, 207, 208, 226/487/684/688, 259/485/487/684,259/485/532, 259/487/684/688, 259/532, 280, 303, 317, 343, 358, 361,440, 478, 485, 485/487, 485/487/532, 485/487/532/684, 485/487/684,485/487/684/688, 485/532, 485/684, 485/684/688, 532, 532/684/688, 611,615, 630, 675, 684, 684/688, 724, 756, and 788, wherein said positionsare numbered with reference to SEQ ID NO:
 2510. 15. The engineeredsucrose synthase of claim 1, wherein said polypeptide sequence of saidengineered sucrose synthase comprises at least one mutation or mutationset at one or more positions selected from8/25/55/95/208/358/440/517/788, 12/45/47, 12/45/47/48/51/136/142/630,12/45/47/51/136/139/630/758, 12/45/47/136/139/142/675/758,12/45/51/136/139/630/675/756/758, 12/45/51/630/756,12/48/51/136/139/758, 12/136/139/142/756/758, 12/136/142, 12/630/756,25/29/208/440, 25/100/154/208/440/517/705/788, 25/517,29/208/361/517/788, 42/198/199/480/532/539/561, 42/198/532/561,42/198/532/561/724, 42/199/480/532/561, 42/259/480/561, 42/259/480/652,42/480/561, 42/480/561/724, 42/561, 47/51/136/756/758, 55,55/410/440/603/788, 55/517, 55/517/788, 70, 70/642, 77/176/487/615/642,95/603, 106/199/539/561/652, 116, 136/139/142, 154/361/440/517/603/788,176, 198/199/480/561, 198/199/480/561/724, 198/199/561/724, 198/480/561,198/480/724, 199/532/539/561/652/724, 259/480, 267/611/642, 280/440/517,380, 480/561/652, 480/561/652/724/764, 480/724, 517, 532/539/561,532/561/724, 603, and 642, wherein said positions are numbered withreference to SEQ ID NO:
 7506. 16. The engineered sucrose synthase ofclaim 1, wherein said polypeptide sequence of said engineered sucrosesynthase comprises at least one mutation or mutation set at one or morepositions selected from 12/45/95/136/139/199/517/630/756,12/45/95/136/756, 12/45/136/139/199/517/603,12/45/136/139/208/603/630/756, 12/45/136/139/517/603/756,12/45/136/139/517/630/642/756, 12/45/136/139/517/756,12/45/136/139/603/756, 12/45/136/139/642/756,12/45/136/176/517/603/630/642, 12/45/136/208/517/630/756,12/45/136/517/603/642/756/789, 12/45/136/517/630/642/756,12/45/136/603/756, 12/45/136/630/642, 12/45/139/176/208/517/603/630,12/45/139/199/208/603, 12/45/139/517/756, 12/45/139/756,12/45/176/603/630/642/756, 12/45/199/208/517/603/630/756,12/45/208/517/603/642/756, 12/95/136/139/517/603/756,12/95/139/517/630/756, 12/95/139/517/642, 12/95/139/630/642,12/95/199/517/642, 12/95/517/630/756, 12/95/630/756, 12/136,12/136/139/176/517/603/630, 12/136/139/176/517/603/756,12/136/139/176/630/756, 12/136/139/176/642, 12/136/139/176/756,12/136/139/199/208/517/603/756, 12/136/139/199/208/517/630/642/756,12/136/139/208/517, 12/136/139/517, 12/136/139/517/603/630/642/756,12/136/139/517/603/630/756, 12/136/139/517/603/756, 12/136/139/517/630,12/136/139/517/630/642, 12/136/139/517/630/642/756, 12/136/139/517/756,12/136/139/603/630, 12/136/139/603/630/642, 12/136/139/603/630/756,12/136/139/603/642/756, 12/136/139/630, 12/136/139/630/642/756,12/136/139/630/756, 12/136/139/642/756, 12/136/176/208/517/603/630/756,12/136/176/517/642, 12/136/176/603/756, 12/136/199/208/517/603/642/756,12/136/199/208/630/642, 12/136/199/517/756, 12/136/208,12/136/208/603/642, 12/136/517/603/630/642, 12/136/517/603/630/756,12/136/517/630, 12/136/517/630/642, 12/136/517/642, 12/136/517/642/756,12/136/517/756, 12/136/603/630/642/756, 12/136/603/630/756,12/136/603/642, 12/136/603/642/756, 12/136/603/756, 12/136/630,12/136/630/756, 12/139/176/517/603/630/756, 12/139/176/630/756,12/139/199/208/642, 12/139/199/517/630/756, 12/139/208/517,12/139/208/642, 12/139/517/603/642/756, 12/139/517/630/642/756,12/139/517/642, 12/139/603/642/756, 12/139/603/756, 12/139/630/642,12/139/630/642/756, 12/139/630/756, 12/176/517/603/630,12/176/517/630/642/756, 12/176/517/756, 12/176/603/630/756,12/176/603/756, 12/199/208/517/642, 12/199/630/642/756, 12/199/642/756,12/199/756, 12/208/517/603/623/630/642, 12/208/603/630/756,12/208/630/756, 12/517/603/630/642/756, 12/517/603/630/756,12/517/603/756, 12/517/630/642/756, 12/517/642/756, 12/603/630/756,12/603/642/756, 12/603/756, 12/630/642/756, 12/630/756,25/176/198/532/539, 116/142/198/434/440,136/139/176/199/208/517/630/642, 136/139/176/517/630/642/756,136/139/199/517/603/756, 136/139/208/517/630/756,136/139/208/603/630/756, 136/139/517/603/630/642/756,136/139/517/603/642/756, 136/139/517/603/756, 136/139/517/756,136/139/603, 136/139/630/642/756, 136/517/756, 136/603/756, 136/630/642,136/630/756, 136/642, 136/642/756, 136/756, 139/199/208/517/630/756,139/199/517/642, 139/208/517/630/642/756, 139/517/603/756,139/517/630/756, 139/642/756, 154/532/652/788, 199/517/603/630/756,208/517/630/642/756, 517/603/630/642/756, 517/630/756, 603/630/756, and603/756, wherein said positions are numbered with reference to SEQ IDNO:
 8420. 17. An engineered polynucleotide encoding at least oneengineered sucrose synthase polypeptide provided in claim
 1. 18. Avector comprising at least one engineered polynucleotide of claim 17.19. A host cell comprising at least one engineered polynucleotide ofclaim
 17. 20. A method for producing at least one engineered sucrosesynthase, comprising culturing the host cell of claim 19, underconditions such that said engineered sucrose synthase variant isproduced by said host cell.
 21. A composition comprising at least oneengineered sucrose synthase of claim
 1. 22. A method for glycosylationof a substrate comprising providing at least one substrate, at least oneengineered glycosyltransferase, and at least one sucrose synthase ofclaim 1, and contacting said substrate with said glycosyltransferase andsucrose synthase under conditions such that said substrate isglycosylated to produce at least one glycosylated product.
 23. Themethod of claim 22, wherein said glycosylated product comprisesrebaudioside M, wherein said substrate comprises rebaudioside D and/orrebaudioside I, and further comprising providing NDP-glucose.
 24. Themethod of claim 22, wherein said glycosylated product comprisesrebaudioside A and/or rebaudioside I, wherein said substrate comprisesat least one stevioside substrate, and further comprising providingNDP-glucose.
 25. The method of claim 22, wherein said glycosylatedproduct comprises rebaudioside D, wherein said substrate comprises atleast one stevioside substrate, and further comprising providingNDP-glucose.
 26. The method of claim 22, wherein said NDP-glucose isselected from ADP-glucose, CDP-glucose, TDP-glucose, GDP-glucose, and/orIDT-glucose.
 27. A rebaudioside produced according to the method ofclaim 22.